Methods, Compounds and Compositions for Repelling Insects and/or Arachnids

ABSTRACT

The present specification discloses natural repellent compounds, and isomeric forms, structural analogues and derivatives of the said compounds, having repellent activity against mosquitoes and other blood-sucking arthropods, compositions comprising such repellent compounds, methods of screening for such repellent compounds, methods of reducing mosquito bites on an individual and methods of reducing mosquito infestation to a location.

This application is a continuation that claims priority to and thefiling date pursuant to 35 U.S.C. §120 of U.S. patent application Ser.No. 14/776,330, filed on Sep. 14, 2015, a 35 U.S.C. §371 U.S. nationalstage entry of International Application PCT/EP2014/055170, filed Mar.14, 2014, that claims the benefit of priority and is entitled to thefiling date of U.S. Provisional Patent Application 61/783,955, filed onMar. 14, 2013, the contents of each of which are hereby incorporated byreference in its entirety.

TECHNICAL FIELD

The present invention relates to novel compounds and compositions, toinsect repellent compounds and compositions, to methods of using thecompounds and compositions, to methods of screening and/or identifyingcompounds and/or compositions for their insect repellent activity.

INTRODUCTION

Mosquito-borne diseases, including malaria, yellow fever, and denguefever, West Nile virus, Eastern equine encephalitis and other illnesses,are a major threat to over 2 billion people world-wide. Integration ofdisease treatment with vector suppression is considered the mosteffective means for disease management. However, strenuous efforts tocontrol such mosquito-borne diseases using this integrated approach overthe last 60 years have proven unsuccessful. One reason underlying theineffectiveness of this approach is the widespread resistance of theparasite to commercially available drugs and the absence of effectivevaccines. Another reason involves resistance to the insecticides used invector control measures and personal protection from mosquito bites.Both the failure of the integrated approach for controllingmosquito-borne diseases and the unavailability of new drugs has led tothe exploration of new directions for vector control measures aimed atdisease eradication.

The most common mosquito repellents available in the market contain DEET(N,N-diethyl-3-methylbenzamide) or Picaridine (1-piperidinecarboxylicacid 2-(2-hydroxyethyl)-1-methylpropylester). DEET is a broad-spectrumrepellent that is effective against mosquitoes and other biting insects.Also a broad-spectrum repellent, Picaridine is a synthetic derivative oflatrou, et al., Methods, Compounds and Compositions for RepellingInsects and/or Arachnids piperine, a compound found in plants used toproduce black pepper. Despite their effectiveness, both of thesesynthetic repellents have many drawbacks. Besides its unpleasant odorand poor skin penetration, DEET elicits allergic reactions and is apossible neurotoxin and carcinogen in mammals. In addition, DEET reactswith certain plastics and synthetic materials, resulting in considerabledamage to eyeglasses and watchbands, and other plastic items. Picaridinehas been associated with both skin and eye irritation. Because of thegeneral public's concern about the safety of these synthetic repellents,there has been increasing need to identify new natural and syntheticcompounds having repellent activity similar to DEET and Picaridine, butlacking their undesirable properties.

The present specification discloses such mosquito repellents and usesand methods for identifying such compounds. The disclosed mosquitorepellents will benefit current approaches being used to controlmosquito-borne diseases.

SUMMARY

The present invention relates to novel compounds and compositions. In anaspect, the invention relates to compounds and compositions forrepelling arthropods, in particular insects and arachnids.

Aspects of the present specification disclose repellent compounds thatbind to specific chemosensory proteins from the chemosensory signalingpathways used by mosquitoes and affect the behavioral activity ofmosquitoes by eliciting an avoidance response. A repellent compounddisclosed herein has mosquito repellent activity and may bind at leastone of the following mosquito odorant-binding proteins (OBPs): OBP1,OBP3, OBP4, OBP5, OBP20, OBP47 and/or other selected OBPs. The disclosedrepellent compounds have a mosquito repellence activity, reduce amosquito-mammalian host interaction, and/or reduce an ability of amosquito to obtain a blood meal from a mammal.

Other aspects of the present specification disclose compositionscomprising a plurality of repellent compounds disclosed herein. Theplurality of repellent compounds includes a carvacrol compound, a cumincompound, a cinnamate compound, or any combination thereof. Compositionsdisclosed herein may, for example, comprise a carvacrol compounddisclosed herein and one or more additional repellent compounds havingmosquito repellent activity. The one or more additional repellentcompounds include a cumin compound, a cinnamate compound, or anycombination thereof. The disclosed compositions have a mosquitorepellence activity, reduce mosquito-mammalian host interaction, and/orreduce an ability of a mosquito to obtain a blood meal from a mammal.

Other aspects of the present specification disclose methods of reducingmosquito bites on an individual, the method comprising the step ofapplying a repellent compound or composition disclosed herein to theindividual, wherein application of the repellent compound or compositionrepels a mosquito from the individual, thereby reducing mosquito bites.Application of the composition may be by direct or indirectadministration.

Other aspects of the present specification disclose methods of reducinga mosquito infestation to a location, the method comprising the steps ofapplying a repellent compound or composition disclosed herein to thelocation, wherein the application repels mosquitoes from the location,thereby reducing the mosquito infestation. The location may be a plantor group of plants, a particular area of land, or a man-made structure,such as, e.g., a commercial building, a house, a shed, or other physicalstructure.

In an aspect, the invention provides a composition comprising acombination of two or more essential oils or essential oil fractions orcombinations thereof as repellents.

In an aspect, the invention relates to novel compounds and compositions,in particular for repelling terrestrial arthropods.

In an aspect, the invention relates to compounds and compositions thatare capable of repelling insects and arachnids that can act as vectorsfor infectious pathogens.

In an aspect, the invention relates to compounds and compositions forpreventing arthropod-borne diseases.

In an aspect, the invention provides a compound of formula (IV) as arepellent of insects and/or arachnids:

wherein R⁴ is a C2-C10, preferably a C3-C10, most preferably a C4-C10aliphatic substituent. The invention also relates to methods forrepelling insects using and/or applying the compound of formula (IV).

In an aspect, the invention provides a method for screening and/oridentifying essential oils having repellent activity with respect toblood-feeding insects and/or arachnid, the method comprising the step ofexposing an essential oil or a fraction thereof to be screened to one ormore odorant-binding protein (OBP) and determining an binding affinityof said oil or fraction with respect to said OBP, wherein an essentialoil or fraction thereof is identified as having insect and/or arachnidrepellent activity if it has a binding affinity to said OBP.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows GC-EAG analysis of four selected essential oils that showaffinity to OBPs and induce repellence. Some of the identified compoundsare indicated in the readouts presented. The female A. gambiae antennawas used as a biological detector (upper trace in each case) for activeconstituents of the essential oils. The lower traces in each case arethe flame ionization detector responses.

FIG. 2 shows the repellence index of compositions comprising differentcompounds in accordance with embodiments of the invention, compared to areference compound (DEET), each compound or combination of compounds(carvacrol+cumin alcohol, and carvacrol+butyl-cinnamate) being appliedat different increasing concentrations as indicated at the bottom of thefigure. Compounds are: A: carvacrol (Cary), B: cumin alcohol (CuAlc), C:ethyl-cinnamate (CinEt), and D: butyl-cinnamate (CinBut).

FIGS. 3 A and 3 B show induced exophily as a measure of repellency ofindividual chemical compounds on Anopheles gambiae s.l. (FIG. 3 A) andCulex spp. (FIG. 3 B), respectively, as compared to DEET, and inducedexophily as a measure of repellence of binary combinations of carvacrolwith either ethyl-cinnamate, butyl-cinnamate or cumin alcohol onAnopheles gambaie s.l. and Culex spp. as compared to DEET. The testedcompounds are carvacrol (Cary), cumin alcohol (CuAlc),(E)-ethyl-cinnamate (CinEt) and butyl-cinnamate (CinBut). ContMeth is amethanol control.

FIGS. 4 A and 4 B are as FIGS. 3A and 3B, but instead of individualcompounds binary composition were tested to compare to DEET.

DESCRIPTION

Insect chemosensory proteins (CSPs) regulate or control crucial insectbehaviors. The chemosensory system consists of several chemosensoryprotein (CSP) classes. Chemosensory protein classes that are importantin the design of novel insect control products include soluble proteinsfound in the antennal sensory lymph and the maxillary palps, such asOBPs and sensory appendage proteins (SAPs). OBPs and SAPs are carrierproteins that facilitate the transport of stimuli from the exterior suchas odor molecules through the aqueous lymph of sensory appendages to thesurfaces of neuronal cells. There, the protein/odorant moleculecomplexes bind odorant receptors (ORs) and initiate a signaling cascadethat results in a behavioral response to the external odour or stimulus.Insects use chemosensory cues from the environment to control criticalbehaviors, such as feeding and mating. Thus, insect chemosensoryproteins are promising targets for the discovery of novel insect controlproducts based on manipulating insect behavior.

Research on insect repellents has identified various plants as potentialsources of essential oils or fumigants that are effective at repellingmosquitoes and other insect pests. These essential oils have a pleasantfragrance, relatively low mammalian toxicity, and a vapor pressuresuitable for action as a volatile spatial repellent. Although one ormore chemical compounds contained in essential oils are responsible fortheir repellence, it is not necessarily true that the most abundantcompounds are responsible for this activity. In addition, the structuresof these compounds cover a very wide diversity of chemical classes andmolecular sizes making it difficult to build a consensus rationalerelating to the repellent activity for these compounds.

The present specification discloses compositions that are active asrepellents of arthropods, in particular terrestrial arthropods, such asinsects and arachnids. In an embodiment, the compositions of theinvention repel insects and arachnids. In embodiment, the composition ofthe invention repels blood-feeding arthropods, in particularblood-feeding insects and/or ticks. In a preferred embodiment, theinvention relates to repellents of insects and arachnids that act asvectors for a pathogen. In an embodiment, the invention relates torepellents of insects and ticks.

The compositions of the invention have been shown to repel one or moreselected from Anopheles, Aedes, and Culex mosquitos, sand flies andixodid ticks. The compositions of the invention are suitable to repelone or more selected from Anopheles gambiae, Aedes aegypti, Lutzomyialongipalpis, and ixodid ticks, such as Ixodes ricinus.

The invention relates to compounds and compositions for preventinginfection of insect-borne and/or tick-borne diseases.

The present specification discloses improved mosquito repellents anduses and methods for identifying such repellents. By realizing thatrepellent compounds could bind to specific chemosensory proteins fromthe chemosensory signaling pathways used by mosquitoes, effectiverepellent compounds have been identified and isolated from essentialoils. In some embodiments, the compounds are bound by OBPs and showmosquito repellent effects. In addition, the present specificationdiscloses various combinations of these repellent compounds that whencombined produce behavioral effects having similar, if not better,repellence to that of DEET. As such, the repellents disclosed hereinmanipulate the mosquito's chemosensory signalling pathway and affect thebehavioral activity of mosquitoes by eliciting an avoidance response.The repellents disclosed herein are intended for agricultural,commercial, and consumer use. For example, the mosquito repellentsdisclosed herein are useful to repel mosquitoes from areas where humansreside in order to reduce the transmission of mosquito-borne diseases.As another non-limiting example, the mosquito repellents disclosedherein can be applied to humans to reduce or prevent mosquitoes fromobtaining a blood-meal from that person. As yet another non-limitingexample, the mosquito repellents disclosed herein are useful to keepaway mosquitoes from outdoor areas where human activities are occurringand would otherwise be disrupted by mosquito presence, such as, e.g., anoutdoor activity like a sporting event or picnic. Similarly, themosquito repellents disclosed herein are useful to keep away mosquitoesfrom naturally occurring or man-made structures containing standingwater in order to prevent egg-laying and mosquito larva development.Other uses of the mosquito repellents disclosed herein are discussedbelow and are readily apparent to a person of ordinary skill.

Mosquitoes are insects of the Order Diptera, Superfamily Culicoidea.Comprising a group of about 3,500 species that live throughout theworld, mosquitoes are divided into three subfamilies (Anophelinae,Culicinae, and Toxorhynchitinae) comprising 41 genera including, withoutlimitation, Anopheles, Aedes, and Culex. Malaria is transmitted byfemale mosquitoes of the genus Anopheles, and of the approximately 430described species of Anopheles over 100 are known to be able to transmitmalaria to humans. Yellow and dengue fever are transmitted by femalemosquitoes from the genus Aedes, while West Nile virus, filariasis,Japanese and St Louis encephalitis and avian malaria are transmitted byfemale mosquitoes from the genus Culex.

Aspects of the present specification disclose, in part a repellentcompound. As used herein, the term “repellent compound” is synonymouswith “mosquito repellent” refers to a compound that binds to an OBPand/or SAP and induces a behavioral response which causes the insect tomove away from the source of the repellent compound and/or reduce orprevent the mosquito's ability to obtain a blood-meal from a mammal. Arepellent compound will typically preferentially bind, withoutlimitation, at least one of the following mosquito OBPs: odorant-bindingprotein 1 (OBP1; SEQ ID NO: 1), odorant-binding protein 3 (OBP3; SEQ IDNO: 2), odorant-binding protein 4 (OBP4; SEQ ID NO: 3), odorant-bindingprotein 5 (GBPS; SEQ ID NO: 4), odorant-binding protein 20 (OBP20; SEQID NO: 5), and/or odorant-binding protein 47 (OBP47; SEQ ID NO: 6). Inother embodiments, a repellent compound does not bind to an OBP.

In an aspect, the invention provides a method of screening, preferablyprescreening essential oils and/or fractions thereof using an OBPbinding assay. This screening or prescreening is an efficient way ofreducing the number of samples to be analyzed in subsequent sampleanalysis and compound identification steps and methods. The presentspecification contains experimental details with respect to theassessment of the OBP binding affinity of candidate essential oils orfractions thereof.

In some embodiments, the method of the invention comprises the step ofexposing a surface mimicking the surface of a mammal to an essential oiland/or fraction thereof or to compositions as disclosed herein anddetermining a number of landings of said insects and/or, more generally,contacts in the case of arachnids on said surface, wherein an essentialoil or fraction thereof or composition as disclosed herein is identifiedas having insect and/or arachnid repellent activity if it is effectiveto reduce the number of landings or contacts by said insects and/orarachnids. Similarly, this method can be used preferably together withOBP assay for prescreening or screening agents, compositions orcompounds having repellence activity. The surface mimicking the surfaceof a mammal is preferably a warm surface (30-34° C.). Preferably, thecarbon dioxide concentration is increased on the surface. A detailedmethodology of such an assay is described in the examples.

Aspects of the present specification disclose, in part a carvacrolcompound. Non-limiting examples of suitable carvacrol compounds include,e.g., carvacrol (5-isopropyl-2-methylphenol) and thymol(2-Isopropyl-5-methylphenol).

In an embodiment, a repellent compound is a carvacrol compound or isselected from carvacrol compounds. In another embodiment, a repellentcompound is a carvacrol compound having a structure of formula I:

wherein R¹ is H, OH, ═O, a halogen, or an optionally substituted alkyl,an alkoxy. In aspects of this embodiment, an optionally substitutedalkyl is an optionally substituted C1-6 alkyl. In other aspects of thisembodiment, a halogen is F, Cl, Br, or I. In aspects of this embodiment,an alkoxy is —OCH₃, —OC₂H₅, —OC₃H₇, or —OC₄H₉.

As used herein the term “alkyl” has the broadest meaning generallyunderstood in the art, and may include a moiety composed of carbon andhydrogen containing no double or triple bonds. Alkyl may be linearalkyl, branched alkyl, cycloalkyl, or a combination thereof, and in someembodiments, may contain from one to thirty-five carbon atoms. Inaspects of this embodiment, alkyl may include C1-10 linear alkyl, suchas methyl (—CH₃), ethyl (—CH₂CH₃), n-propyl (—CH₂CH₂CH₃), n-butyl(—CH₂CH₂CH₂CH₃), n-pentyl (—CH₂CH₂CH₂CH₂CH₃), n-hexyl(—CH₂CH₂CH₂CH₂CH₂CH₃), etc.; C₃₋₁₀ branched alkyl, such as C₃H₇ (e.g.iso-propyl), C₄H₉ (e.g. branched butyl isomers), C₅H₁₁ (e.g. branchedpentyl isomers), C₆H₁₃ (e.g. branched hexyl isomers), C₇H₁₅ (e.g. heptylisomers), etc.; C₃₋₁₀ cycloalkyl, such as C₃H₅ (e.g. cyclopropyl), C₄H₇(e.g. cyclobutyl isomers such as cyclobutyl, methylcyclopropyl, etc.),C₅H₉ (e.g. cyclopentyl isomers such as cyclopentyl, methylcyclobutyl,dimethylcyclopropyl, etc.) C₆H₁₁ (e.g. cyclohexyl isomers), C₇H₁₃ (e.g.cycloheptyl isomers), etc.; and the like.

With respect to an optionally substituted moiety such as optionallysubstituted alkyl, a phrase such as “optionally substituted alkyl”refers to an alkyl that may be unsubstituted, or may have one or moresubstituents, and does not limit the number of carbon atoms in anysubstituent. A phrase such as “C₁₋₁₂ optionally substituted alkyl”refers to unsubstituted C₁₋₁₂ alkyl, or substituted alkyl wherein boththe alkyl parent and all substituents have from 1-12 carbon atoms.Similar conventions may be applied to other optionally substitutedmoieties such as aryl and heteroaryl.

Substituents on alkyl may be the same as those described generallyabove, except that alkyl may not have an alkyl substituent. In someembodiments, substituents on alkyl are independently selected from F,Cl, Br, I, OH, NH, ═O, etc.

As used herein, the term “alkoxy” includes —O-alkyl, such as —OCH₃,—OC₂H₅, —OC₃H₇ (e.g. propoxy isomers such as isopropoxy, n-propoxy,etc.), —OC₄H₉ (e.g. butyoxy isomers), —OC₅H₁₁ (e.g. pentoxy isomers),—OC₆H₁₃ (e.g. hexoxy isomers), —OC₇H₁₅ (e.g. heptoxy isomers), etc.

In aspects of this embodiment, a carvacrol compound is one of thefollowing compounds:

Aspects of the present specification disclose, in part a cumin compoundand/or cumin compounds. Non-limiting examples of suitable cumincompounds include, e.g., cumin alcohol, cumin aldehyde and cuminic acid.

In an embodiment, a repellent compound is a cumin compound. In anotherembodiment, a repellent compound is a cumin compound having a structureof formula II:

wherein R² is H, OH, ═O, a halogen, a carbonyl group (CHO), a carboxylgroup (OOH), or an optionally substituted alkyl, an alkoxy. In aspectsof this embodiment, an optionally substituted alkyl is an optionallysubstituted C₁₋₆ alkyl. In other aspects of this embodiment, a halogenis F, Cl, Br, or I. In aspects of this embodiment, an alkoxy is —OCH₃,—OC₂H₅, —OC₃H₇, or —OC₄H₉.

In aspects of this embodiment, a cumin compound is one of the followingcompounds:

Aspects of the present specification disclose, in part a cinnamatecompound and/or cinnamate compounds. Non-limiting examples of suitablecinnamate compounds include, e.g., cinnamate[(E)-3-phenylprop-2-enoate], methyl cinnamate (methyl3-phenylprop-2-enoate), ethyl cinnamate (ethyl 3-phenylprop-2-enoate),butyl cinnamate (butyl 3-phenylprop-2-enoate), isobutyl-cinnamate(isobutyl 3-phenylprop-2-enoate), N-butyl-cinnamate (N-butyl3-phenylprop-2-enoate), isopropyl-cinnamate (isopropyl3-phenylprop-2-enoate), E-cinnamyl acetate, cinnamaldehyde[(2E)-3-phenylprop-2-enal], E-cinnamaldehyde, Z-cinnamaldehyde, ando-methoxycinnamaldehyde.

In an embodiment, a repellent compound is a cinnamate compound. Inanother embodiment, a repellent compound is a cinnamate compound havinga structure of formula III:

wherein R³ is H, OH, ═O, a halogen, or an optionally substituted alkyl,an alkoxy. In aspects of this embodiment, an optionally substitutedalkyl is an optionally substituted C₁₋₆ alkyl. In other aspects of thisembodiment, a halogen is F, Cl, Br, or I. In aspects of this embodiment,an alkoxy is —OCH₃, —OC₂H₅, —OC₃H₇, or —OC₄H₉.

In another embodiment, a repellent compound is a cinnamate compoundhaving a structure of formula IV:

wherein R⁴ is H, OH, ═O, CH₃, a halogen, or an optionally substitutedalkyl, an alkoxy. In aspects of this embodiment, an optionallysubstituted alkyl is an optionally substituted C₁₋₆ alkyl. In otheraspects of this embodiment, a halogen is F, Cl, Br, or I. In aspects ofthis embodiment, an alkoxy is —OCH₃, —OC₂H₅, —OC₃H₇, or —OC₄H₉.

In a preferred embodiment, R⁴ is a C1-C10 aliphatic substituent,preferably a C2-C10 aliphatic substituent. In a preferred embodiment, R⁴is a C1-C8 aliphatic substituent, preferably a C2-C8 aliphaticsubstituent. In a preferred embodiment, R⁴ is a C1-C7 aliphaticsubstituent, preferably a C2-C7 aliphatic substituent, and mostpreferably, R⁴ is a C1-C6 aliphatic substituent, preferably a C2-C6aliphatic substituent. In an embodiment, R⁴ is a C3-C10, preferablyC3-C8, more preferably C3-C7 and most preferably C3-C6 aliphaticsubstituent.

For example, said aliphatic substituent is a substituted orunsubstituted alkyl or alkenyl.

Preferably, R⁴ is a C1-C10, preferably C2-C10 alkyl. For example, R⁴ isa C1-C7, preferably a C2-C7 alkyl. More preferably, R⁴ is a C1-C10,preferably a C2-C6 alkyl.

In a preferred embodiment, R⁴ is a C2-C10, preferably C3-C10 alkyl. Forexample, R⁴ is a C2-C7, preferably a C3-C7 alkyl. More preferably, R⁴ isa C2-C10, preferably a C3-C6 alkyl.

Cinnamate compounds having C2-C10, preferably C3-C10 aliphaticsubstituent were not found in essential oils but were surprisingly foundto have high insect and arachnid repellency activity. Surprisingly,these cinnamate derivatives were found to have even higher insect and/orarachnid repellence activity than methyl cinnamate, the cinnamatecompound that is naturally occurring in some essential oils. Therefore,the invention encompasses compositions comprising synthetic or naturalcompounds, for example compositions with different cinnamate compounds.

In aspects of this embodiment, a cinnamate compound is one of thefollowing compounds:

Aspects of the present specification provide, in part, a compositioncomprising a mosquito repellent disclosed herein. A compositiondisclosed herein comprises a repellent compound disclosed herein and isuseful in repelling insects and/or arachnids, such as mosquitoes andticks from an individual and/or a location treated with the composition.As such, a composition disclosed herein is useful for any applicationthat reduces mosquito vector human host and/or animal host interactions.A composition may be administered to an individual alone, or incombination with other supplementary active ingredients, agents, ordrugs.

A composition disclosed herein may comprise one or more repellentcompounds disclosed herein. In one embodiment, a composition disclosedherein may comprise only a single repellent compound disclosed herein.In another embodiment, a composition disclosed herein may comprise aplurality of repellent compounds disclosed herein. In aspects of thisembodiment, a composition disclosed herein comprises at least tworepellent compounds, at least three repellent compounds, at least fourrepellent compounds, or at least five repellent compounds. In otheraspects of this embodiment, a composition disclosed herein comprises atmost two repellent compounds, at most three repellent compounds, or atmost four repellent compounds. In yet other aspects of this embodiment,a composition disclosed herein comprises one to three repellentcompounds, two to four repellent compounds, two to five repellentcompounds, three to five repellent compounds, or two to three repellentcompounds. In aspects of this embodiment, a repellent compound includes,without limitation, a carvacrol compound disclosed herein, a cinnamatecompound disclosed herein, a cumin compound disclosed herein, or anycombination thereof.

In an embodiment, the composition of the invention comprises acombination of two or more compounds having insect and/or arachnidrepellent activity, said two or more compounds being selected,independently, from carvacrol compounds, cumin compounds, and cinnamatecompounds.

In a preferred embodiment, the composition comprises at least onecarvacrol compound. For example, the composition comprises carvacrol orthymol and one or more other insect repellent compounds disclosedherein. In an embodiment, the composition comprises carvacrol and thymol(two carvacrol compounds) and one or more other insect repellentcompounds disclosed herein, for example one or more cumin compoundsand/or one or more cinnamate compounds.

In an embodiment, the composition comprises carvacrol and one or moreselected from the group consisting of thymol, a cumin compound and acinnamate compound.

In an embodiment, the composition of the invention comprises at least acarvacrol compound and one or more selected from the group of a cumincompound and a cinnamate compound. Preferably, the composition comprisescarvacrol and one or more selected from the group of a cumin compoundand a cinnamate compound.

In an embodiment, the composition comprises carvacrol and one or morecumin compounds, for example one or more selected from cumin alcohol,cumin aldehyde and cuminic acid. In accordance with a preferredembodiment, the composition comprises carvacrol and one or more cumincompounds selected from cumin alcohol and cuminic acid.

Alternatively, the composition comprises thymol and one or more cumincompounds, for example one or more selected from cumin alcohol, cuminaldehyde and cuminic acid, preferably from cumin alcohol and cuminicacid.

In an embodiment, the composition comprises carvacrol and cumin alcohol.In a preferred embodiment, the composition comprises carvacrol andcuminic acid.

In an embodiment, the composition comprises carvacrol and one or morecinnamate compounds, for example carvacrol and one or more selected frommethyl cinnamate and one or more cinnamate compounds of formula (IV)with R⁴ being a C2-C10, preferably C3-C10 aliphatic substituent, forexample a C2-C10 or C3-C10 alkyl, in accordance with the preferredembodiment specified herein above.

Alternatively, the composition comprises thymol and one or more selectedfrom methyl cinnamate and one or more a cinnamate compound of formula(IV) with R⁴ being a C2-C10, preferably C3-C10 aliphatic substituent,for example a C2-C10, preferably C3-C10 alkyl, in accordance with thepreferred embodiment specified herein above.

In an embodiment, the composition comprises carvacrol and one or moreC2-C10 alkyl cinnamate, preferably C2-C6 alkyl cinnamate, preferablyethyl- and/or butyl cinnamate.

In a preferred embodiment, the composition comprises carvacrol and oneor more C3-C10 alkyl cinnamates, preferably a C3-C6 alkyl cinnamate, forexample butyl-cinnamate.

In an embodiment, the composition comprises a carvacrol compound, acumin compound and a cinnamate compound. For example, the compositioncomprises carvacrol, a cumin compound and a cinnamate compound.Alternatively, the composition comprises thymol, a cumin compound and acinnamate compound.

In an embodiment, the composition comprises carvacrol, one or more cumincompounds selected from cumin alcohol, cumin aldehyde and cuminic acid,and one or more cinnamate compounds selected from methyl cinnamate andone or more cinnamate compounds of formula (IV) with R⁴ being a C2-C10,preferably C3-C10 aliphatic substituent, for example a C2-C10 alkyl,preferably C2-C6 alkyl, more preferably a C3-C10 alkyl, such as a C3-C6alkyl.

In a preferred embodiment, the composition comprises carvacrol, one ormore cumin compounds selected from cumin alcohol, cumin aldehyde andcuminic acid, and one or more cinnamate compounds selected fromcompounds of formula (IV) with R⁴ being a C2-C10, preferably C3-C10aliphatic substituent, for example a C2-C10 or C3-C10 alkyl, preferablyC2-C6 or C3-C6 alkyl, preferably from ethyl and butyl cinnamate.

In another embodiment, the composition comprises thymol, one or morecumin compounds selected from cumin alcohol, cumin aldehyde and cuminicacid, and one or more cinnamate compounds selected from methyl cinnamateand one or more cinnamate compounds of formula (IV) with R⁴ being aC2-C10 aliphatic substituent, for example a C2-C10 alkyl, preferablyC2-C6 alkyl. In an embodiment, R4 is a C3-C10 aliphatic substituent, forexample a C3-C10 alkyl, preferably C3-C6 alkyl.

In an embodiment, the composition comprises carvacrol, one or more cumincompounds selected from cumin alcohol, cumin aldehyde and cuminic acid,and one or more cinnamate compounds selected from C2-C10 or C3-C10alkyl, preferably C2-C6 or C3-C6 alkyl cinnamates.

In an embodiment, the composition comprises carvacrol, one or more cumincompounds selected from cumin alcohol, cumin aldehyde and cuminic acid,and one or more cinnamate compounds selected from ethyl and butylcinnamate.

In a preferred embodiment, the composition comprises carvacrol, oneselected from cuminic acid and cumin alcohol and one selected from ethylcinnamate and butyl cinnamate.

In an embodiment, the composition comprises a cumin compound and acinnamate compound. In an embodiment, the composition comprises one ormore selected from the group consisting of cuminic acid, cumin alcoholand cumin aldehyde, and one or more selected from the group of methylcinnamate, methyl cinnamate and one or more cinnamate compounds offormula (IV) with R⁴ being a C2-C10 aliphatic substituent, for example aC2-C10 alkyl, preferably C2-C6 alkyl, preferably a C3-C10 aliphaticsubstituent, for example a C3-C10 alkyl, preferably C3-C6 alkyl.

In an embodiment, the composition comprises one or more selected fromthe group consisting of cuminic acid, cumin alcohol and cumin aldehyde,and one or more selected from cinnamate compounds of formula (IV) withR⁴ being a C2-C10 aliphatic substituent, for example a C2-C10 alkyl,preferably C2-C6 alkyl. In an embodiment, R4 is a C3-C10 aliphaticsubstituent, for example a C3-C10 alkyl, preferably C3-C6 alkyl.

In an embodiment, the composition comprises one or more selected fromthe group consisting of cuminic acid, cumin alcohol and cumin aldehyde,and one or more selected from C2-C10, preferably C2-C6 alkyl cinnamates.Preferably, said cinnamate is a C3-C10, preferably a C3-C6 cinnamate.

In an embodiment, the composition comprises cumin aldehyde and one ormore selected from C1-C10, preferably C2-C6 alkyl cinnamates. In anembodiment, the composition comprises cuminic acid and one or moreselected from C1-C10, preferably C2-C6 alkyl cinnamates. In anembodiment, the composition comprises cumin alcohol and one or moreselected from C1-C10, preferably C2-C6 alkyl cinnamates. Preferably,said alkyl is C3-C10, more preferably C3-C6 alkyl cinnamates.

In an embodiment, the composition of the invention comprises twodifferent cumin compounds. Preferably, the composition comprises two ormore selected from the group consisting of cuminic acid, cumin alcoholand cumin aldehyde. For example, the composition comprises cuminic acidand cumin aldehyde. In an embodiment, the composition comprises cuminicacid and cumin alcohol. In an embodiment, the composition comprisescumin aldehyde and cumin alcohol. In an embodiment, the compositioncomprises cuminic acid, cumin aldehyde and cumin alcohol.

In an embodiment, the composition comprises two or more cinnamatecompounds. In an embodiment, the composition comprises methyl cinnamate,ethyl cinnamate and one or more cinnamate compounds of formula (IV) withR⁴ being a C2-C10, in particular a C3-C10 aliphatic substituent, forexample a C2-C10 alkyl, for example C3-C10 alkyl, preferably C2-C6alkyl, more preferably C3-C6 alkyl. In an embodiment, the compositioncomprises two different cinnamate compounds selected from compounds offormula (IV) with R⁴ being a C2-C10 aliphatic substituent, for example aC2-C10 alkyl, preferably C2-C6 alkyl.

In a preferred embodiment, the composition comprises two differentcinnamate compounds selected from compounds of formula (IV) with R⁴being a C3-C10 aliphatic substituent, for example a C3-C10 alkyl,preferably C3-C6 alkyl.

In an embodiment, the composition of the invention comprises a carvacrolcompound and a cinnamate compound. Preferably, the composition comprisescarvacrol and one, two or more cinnamate compounds.

In an embodiment, the composition of the invention comprises a carvacrolcompound and a cumin compound. Preferably, the composition comprisescarvacrol and one, two or more cumin compounds.

In an embodiment, a composition disclosed herein includes a carvacrolcompound. In aspects of this embodiment, a composition comprises,carvacrol, thymol, or any combination thereof.

In another embodiment, a composition disclosed herein includes acarvacrol compound and a cumin compound. In aspects of this embodiment,a composition comprises cumin alcohol, cumin aldehyde, cuminic acid, orany combination thereof.

In another embodiment, a composition disclosed herein includes acarvacrol compound and a cinnamate compound. In aspects of thisembodiment, a composition comprises cinnamate, methyl cinnamate, ethylcinnamate, butyl cinnamate, isobutyl-cinnamate, N-butyl cinnamate,isopropyl cinnamate, E-cinnamyl acetate, cinnamaldehyde,E-cinnamaldehyde, Z-cinnamaldehyde, o-methoxycinnamaldehyde, or anycombination thereof.

In another embodiment, a composition disclosed herein comprises acarvacrol compound disclosed herein and a single additional repellentcompound disclosed herein. In another embodiment, a compositiondisclosed herein comprises a carvacrol compound disclosed herein and oneor more additional repellent compounds disclosed herein. In aspects ofthis embodiment, a composition disclosed herein comprises a carvacrolcompound and at least one additional repellent compound, at least twoadditional repellent compounds, at least three additional repellentcompounds, at least four additional repellent compounds. In otheraspects of this embodiment, a composition disclosed herein comprises acarvacrol compound and at most one additional repellent compound, atmost two additional repellent compounds, at most three additionalrepellent compounds, at most four additional repellent compounds. In yetother aspects of this embodiment, a pharmaceutical composition disclosedherein comprises a carvacrol compound and one to three additionalrepellent compounds, two to four additional repellent compound, two tothree additional repellent compounds, two to five additional repellentcompound, or three to five additional repellent compound. In aspects ofthis embodiment, an additional repellent compound includes, withoutlimitation, a cinnamate compound disclosed herein, a cumin compounddisclosed herein, or any combination thereof.

In another embodiment, a composition disclosed herein comprises acinnamate compound disclosed herein and a single additional repellentcompound disclosed herein. In another embodiment, a compositiondisclosed herein comprises a cinnamate compound disclosed herein and oneor more additional repellent compounds disclosed herein. In aspects ofthis embodiment, a composition disclosed herein comprises a cinnamatecompound and at least one additional repellent compound, at least twoadditional repellent compounds, at least three additional repellentcompounds, at least four additional repellent compounds. In otheraspects of this embodiment, a composition disclosed herein comprises acinnamate compound and at most one additional repellent compound, atmost two additional repellent compounds, at most three additionalrepellent compounds, at most four additional repellent compounds. In yetother aspects of this embodiment, a pharmaceutical composition disclosedherein comprises a cinnamate compound and one to three additionalrepellent compounds, two to four additional repellent compound, two tothree additional repellent compounds, two to five additional repellentcompound, or three to five additional repellent compound. In aspects ofthis embodiment, an additional repellent compound includes, withoutlimitation, a carvacrol compound disclosed herein, a cumin compounddisclosed herein, or any combination thereof.

In another embodiment, a composition disclosed herein comprises a cumincompound disclosed herein and a single additional repellent compounddisclosed herein. In another embodiment, a composition disclosed hereincomprises a cumin compound disclosed herein and one or more additionalrepellent compounds disclosed herein. In aspects of this embodiment, acomposition disclosed herein comprises a cumin compound and at least oneadditional repellent compound, at least two additional repellentcompounds, at least three additional repellent compounds, at least fouradditional repellent compounds. In other aspects of this embodiment, acomposition disclosed herein comprises a cumin compound and at most oneadditional repellent compound, at most two additional repellentcompounds, at most three additional repellent compounds, at most fouradditional repellent compounds. In yet other aspects of this embodiment,a pharmaceutical composition disclosed herein comprises a cumin compoundand one to three additional repellent compounds, two to four additionalrepellent compound, two to three additional repellent compounds, two tofive additional repellent compound, or three to five additionalrepellent compound. In aspects of this embodiment, an additionalrepellent compound includes, without limitation, a carvacrol compounddisclosed herein, a cinnamate compound disclosed herein, or anycombination thereof.

In another embodiment, a composition disclosed herein includes acarvacrol compound disclosed herein, a cinnamate compound disclosedherein, and a cumin compound disclosed herein.

In another embodiment, a composition disclosed herein comprises acinnamate compound and one or more additional repellent compoundsdisclosed herein, wherein the one or more additional repellent compoundsdoes not include a carvacrol compound disclosed herein. In an aspect ofthis embodiment, a composition disclosed herein comprises a cinnamatecompound and one or more additional repellent compounds, wherein the oneor more additional repellent compounds does not include a cumincompound.

In another embodiment, a composition disclosed herein comprises a cumincompound and one or more additional repellent compounds disclosedherein, wherein the one or more additional repellent compounds does notinclude a carvacrol compound disclosed herein. In an aspect of thisembodiment, a composition disclosed herein comprises a cumin compoundand one or more additional repellent compounds, wherein the one or moreadditional repellent compounds does not include a cinnamate compound.

In another embodiment, a composition disclosed herein comprises acarvacrol compound and one or more additional repellent compoundsdisclosed herein, wherein the one or more additional repellent compoundsdoes not include a cinnamate compound disclosed herein. In an aspect ofthis embodiment, a composition disclosed herein comprises a carvacrolcompound and one or more additional repellent compounds, wherein the oneor more additional repellent compounds does not include a cumincompound.

In another embodiment, a composition disclosed herein comprises acinnamate compound and a cumin compound and one or more additionalrepellent compounds disclosed herein, wherein the one or more additionalrepellent compounds do not include a carvacrol compound disclosedherein. In an aspect of this embodiment, a composition disclosed hereincomprises a cumin compound and one or more additional repellentcompounds, wherein the one or more additional repellent does neitherinclude a carvacrol compound nor a cinnamate compound

For making the composition in the invention, it is possible to add,independently, one, two or more compounds having insect and/or arachnidrepellent activity as isolated and/or synthetic compounds to saidcomposition. It is also possible to add one or more compounds in theform of one or more essential oils, fraction or concentrate thereof,wherein said compound is comprised in said essential oil, fraction orconcentrate.

Practically, most compounds of the compositions of the invention arereadily available in the form of essential oils comprising the compoundsor as isolates from such essential oils. Cinnamate compounds of formula(IV) with R⁴ being a C3-C10 aliphatic substituent may not all so far beknown from essential oils and may thus preferably be prepared by atleast one chemical synthesis step, for example using cinnamate obtainedfrom essential oils. Of course, also compounds that can be isolated fromessential oils may be obtained by synthesis. Furthermore, preparation ofcompounds my biotechnological means and/or by an overall processincluding one or more steps including isolation from an essential oil,biotechnological conversion and/or synthesis can be employed. Thepresent invention is not intended to be limited to any particular way ofobtaining the compounds disclosed in this specification.

The composition of the invention may be based on compounds or fractionsof extracts isolated from plant that are combined in accordance with theinvention. In a preferred embodiment, the composition of the inventioncomprises or consists of a combination of two or more essential oils oressential oil fractions.

In an embodiment, the composition of the invention comprises one or moreselected from the groupings: (i) a combination of two (or more)different essential oils, (ii) a combination of an essential oil with afraction of a different essential oil, (iii) a combination of twofractions from two different essential oils, and (iv) two differentfractions of the same essential oil.

In an aspect, the invention provides a composition comprising acombination of two or more different essential oils and fractionsthereof, wherein said essential oils are selected from the groupconsisting of: essential oil (EO) of (1) the aerial plat parts ofCoridothymus capitatus (EO 5), (2) aerial plat parts of Origanummajorana (EO 6), (3) leaves of Origanum heracleoticum (EO 9), (4)flowers of Origanum vulgare (EO 14) and (5) leaves of Origanum sp. (EO169).

In an embodiment, the composition of the invention is an insect and/orarachnid repellent composition. Preferably, the composition has aninsect and/or arachnid repellence activity. In an embodiment, acomposition disclosed herein reduces insect and/or arachnid mammalianhost interactions In an embodiment, a composition disclosed hereinreduces the ability of an insect and/or arachnid to obtain a blood-mealfrom a mammal. Preferably, said insect and/or arachnid is selected froma blood-feeding insect and/or arachnid, for example selected frommosquitos, sand flies and ticks.

The composition of the invention can be used to repel insects fromhumans and animals, and reduces arthropod-host interactions in general,not only with respect to humans. An individual, for the purpose of thisspecification, may be a human or animal individual, for example alivestock or companion animal individual. Mammals, for the purpose ofthis specification, include humans and/or animals, in particular humansand mammalian animals, such as mammalian pets and livestock animals, forexample. Animals include also non-mammalian animals, in particularnon-mammalian livestock animals and/or pets, such as birds, poultry, andso forth.

In aspects of this embodiment, presence of a composition (1) repelsinsect and/or arachnid (2) reduces insect and/or arachnid mammalian hostinteractions and/or (3) reduces the ability of an insect and/or arachnidto obtain a blood-meal from a mammal by, e.g., at least 10%, at least15%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, or at least 90%, as compared to nothaving the composition present. In other aspects of this embodiment,presence of a composition (1) repels insects and/or arachnids; (2)reduces insect and/or arachnid mammalian host interactions; and/or (3)reduces an ability of an insect and/or arachnid to obtain a blood-mealfrom a mammal by, e.g., about 10% to about 100%, about 30% to about100%, about 50% to about 100%, about 70% to about 100%, about 20% toabout 90%, about 40% to about 90%, about 60% to about 90%, about 10% toabout 80%, about 30% to about 80%, about 50% to about 80%, about 10% toabout 70%, about 30% to about 70%, or about 50% to about 70%, ascompared to not having the composition present.

A composition disclosed herein can take any of a variety of dosage formsincluding, without limitation, a liquid composition, such as, e.g., asolution, suspension, emulsion; a semi-solid composition, such as, e.g.,an ointment, cream, balm, foam, gel, or salve or a solid composition,such as, e.g., lyophilisate, powder, granule, pellet, capsule; or anyother dosage form suitable for applying a repellent compound/compositiondisclosed herein to a location to be treated. In one embodiment, inliquid, semi-solid, and solid forms, an amount of a repellent compounddisclosed herein typically is between about 0.0001% (w/v) to about 50%(w/v), about 0.001% (w/v) to about 10.0% (w/v), or about 0.01% (w/v) toabout 1.0% (w/v). In another aspect embodiment, in liquid, semi-solid,and solid forms, an amount of a repellent compound disclosed hereintypically is applied between about 0.001 μg/cm² to about 500 μg/cm²,about 0.01 μg/cm² to about 100 μg/cm², or about 0.1 μg/cm² to about 10μg/cm². In another aspect embodiment, in liquid, semi-solid, and solidforms, an amount of a repellent compound disclosed herein typically isbetween about 0.01 nmole/cm² to about 1000 nmole/cm², about 0.1nmole/cm² to about 100 nmole/cm², or about 1 nmole/cm² to about 50nmole/cm². In another embodiment, in liquid, semi-solid, and solidforms, an amount of a repellent compound disclosed herein is typicallybetween about 0.001 mg/L to about 500 mg/L, about 0.01 mg/L to about 100mg/L, or about 0.1 mg/L to about 50 mg/L.

It is noted in general terms that the skilled person will understandthat the concentration of the insect repellent compounds in thecomposition of the invention will be adjusted in accordance with thetype of formulation (slow, fast release, excipients), the physicallocation such as on skin, animal fur, clothing or in a room, thetemperature and/or humidity conditions of the location, and the producttype. The composition of the invention may in particular be used indiverse products, including, for example, topically administeredproducts, compositions for use in connection with an evaporation devicesuch dispensing from a wick, sprays, just to mention a few, as disclosedelsewhere in this specification.

In another embodiment, a composition disclosed herein comprises, whenapplied, about 0.01 μg/cm² to about 50 μg/cm² of a carvacrol compounddisclosed herein, about 0.01 μg/cm² to about 50 μg/cm² of a cumincompound disclosed herein, and/or about 0.01 μg/cm² to about 50 μg/cm²of a cinnamate compound disclosed herein. In aspects of this embodiment,a composition disclosed herein comprises when applied, about 0.1 μg/cm²to about 10 μg/cm² of a carvacrol compound disclosed herein, about 0.1μg/cm² to about 10 μg/cm² of a cumin compound disclosed herein, and/orabout 0.1 μg/cm² to about 10 μg/cm² of a cinnamate compound disclosedherein. In aspects of this embodiment, a composition disclosed hereincomprises, when applied, about 0.5 μg/cm² to about 5 μg/cm² of acarvacrol compound disclosed herein, about 0.5 μg/cm² to about 5 μg/cm²of a cumin compound disclosed herein, and/or about 0.5 μg/cm² to about 5μg/cm² of a cinnamate compound disclosed herein.

In another embodiment, a composition disclosed herein comprises, whenapplied, about 0.01 nmole/cm² to about 100 nmole/cm² of a carvacrolcompound disclosed herein, about 0.01 nmole/cm² to about 100 nmole/cm²of a cumin compound disclosed herein, and/or about 0.01 nmole/cm² toabout 100 nmole/cm² of a cinnamate compound disclosed herein. In aspectsof this embodiment, a composition disclosed herein comprises, whenapplied, about 0.1 nmole/cm² to about 50 nmole/cm² of a carvacrolcompound disclosed herein, about 0.1 nmole/cm² to about 50 nmole/cm² ofa cumin compound disclosed herein, and/or about 0.1 nmole/cm² to about50 nmole/cm² of a cinnamate compound disclosed herein. In aspects ofthis embodiment, a composition disclosed herein comprises, when applied,about 1 nmole/cm² to about 30 nmole/cm² of a carvacrol compounddisclosed herein, about 1 nmole/cm² to about 30 nmole/cm² of a cumincompound disclosed herein, and/or about 1 nmole/cm² to about 30nmole/cm² of a cinnamate compound disclosed herein. In aspects of thisembodiment, a composition disclosed herein comprises, when applied,about 2.5 nmole/cm² to about 25 nmole/cm² of a carvacrol compounddisclosed herein, about 2.5 nmole/cm² to about 25 nmole/cm² of a cumincompound disclosed herein, and/or about 2.5 nmole/cm² to about 25nmole/cm² of a cinnamate compound disclosed herein.

A composition disclosed herein may optionally comprise additionalcomponents such as, e.g., an adhesive, a solvent, a wetting agent, anemulsifying agent, a carrier, a diluent, or a dispersing agent. Suchadditional components are known to a person of skill in the art.

A composition disclosed herein may optionally comprise an additionalmosquito repellent. Non-limiting examples of an additional mosquitorepellent, include, e.g., DEET, ethyl-butylacetylaminopropionate(EBAAP), and Picaridine.

A composition disclosed herein may optionally comprise an insecticide.Non-limiting examples of an insecticide include a organochlorine, suchas, e.g., Aldrin, Chlordane, Chlordecone, DDT, Dieldrin, Endosulfan,Endrin, Heptachlor, Hexachlorobenzene, Lindane(gamma-hexachlorocyclohexane), Methoxychlor, Mirex, Pentachlorophenol,and TDE; an organophosphate, such as, e.g., Acephate, Azinphos-methyl,Bensulide, Chlorethoxyfos, Chlorpyrifos, Chlorpyriphos-methyl, Diazinon,Dichlorvos (DDVP), Dicrotophos, Dimethoate, Disulfoton, Ethoprop,Fenamiphos, Fenitrothion, Fenthion, Fosthiazate, Malathion,Methamidophos, Methidathion, Mevinphos, Monocrotophos, Naled, Omethoate,Oxydemeton-methyl, Parathion, Parathion-methyl, Phorate, Phosalone,Phosmet, Phostebupirim, Phoxim, Pirimiphos-methyl, Profenofos, Terbufos,Tetrachlorvinphos, Tribufos, and Trichlorfon; a carbamate, such as,e.g., Aldicarb, Bendiocarb, Carbofuran, Carbaryl, Dioxacarb, Fenobucarb,Fenoxycarb, Isoprocarb, Methomyl, and 2-(1-Methylpropyl)phenylmethylcarbamate; a pyrethroid, such as, e.g., Allethrin, Bifenthrin,Cyhalothrin, λ-Cyhalothrin, Cypermethrin, Cyfluthrin, Deltamethrin,Etofenprox, Fenvalerate, Permethrin, Phenothrin, Prallethrin,Resmethrin, Tetramethrin, Tralomethrin, and Transfluthrin; and aneonicotinoid, such as, e.g., Acetamiprid, Clothianidin, Imidacloprid,Nitenpyram, Nithiazine, Thiacloprid, and Thiamethoxam.

Aspects of the present specification disclose a method of reducing orpreventing bites by mosquitoes and other blood-sucking arthropods on anindividual animal or human by applying a repellent compound orcomposition disclosed herein to the individual, wherein such applicationof the repellent compound or composition repels a mosquito or otherarthropod from the individual, thereby reducing or preventing bites fora blood-meal. In one embodiment, a repellent compound or compositiondisclosed herein is applied to an individual in order to repel amosquito or other blood-sucking arthropod from obtaining a blood-mealfrom the individual treated. An individual may be any mammal. In anaspect of this embodiment, a mammal is a human being. Application of thecompound or composition may be administered topically using, e.g., alotion, an oil, an ointment, a cream, a balm, a foam, a gel, or salve.

Aspects of the present specification disclose a method of reducing orpreventing a mosquito infestation to a location by applying a repellentcompound or composition disclosed herein to the location, wherein suchapplication repels mosquitoes or other blood-sucking arthropods from thelocation, thereby reducing or preventing the mosquito infestation. Inanother embodiment, the disclosed method is a method of treating anatural area by applying a repellent compound or composition disclosedherein, wherein such application repels a mosquito or otherblood-sucking arthropod from foraging for a blood-meal in the vicinityof the treated natural area. Non-limiting examples of a natural area,include, e.g., a park area, a forested area, an area containing foliage,a pond area or any other area containing standing water. In anotherembodiment, the disclosed method is a method of treating a man-madestructure by applying a repellent compound disclosed herein, whereinsuch application repels a mosquito from foraging for a blood-meal in thevicinity of the treated structure. Non-limiting examples of a man-madestructure include, e.g., a building or part thereof such as a room, abalcony or terrace, a pool, a recreational area, a maintenance space fordomestic animals, for example an incubator-maintenance space for birdsincluding chicken.

As used herein, the term “location” refers to any site to which movementof a mosquito or other blood-sucking arthropod is to be retarded. Alocation includes, by way of example, a plant or group of plants, aparticular area of land, or a man-made structure, such as, e.g., acommercial building, a house, a shed, other physical structure, or partthereof. As used herein, the term “plant” refers to any living organismbelonging to the Kingdom Plantae. Non-limiting examples include trees,flowering plant, herbs, bushes, grasses, vines, ferns, mosses, and greenalgae.

A repellent compound or composition disclosed herein is applied to alocation by any method that can dispense to a location an amount ofrepellent compound effective in repelling a mosquito or otherblood-sucking arthropod. A method of application is not critical andmany well known methods can be used.

In one embodiment, an appropriate amount of a repellent compound orcomposition disclosed herein can be dissolved into an appropriatecompatible solvent and dispensed as a solution into or onto the intendedlocation. The solvent employed is typically a volatile solvent (i.e.,having a boiling point of about 100° C. or less) that will evaporateover a period of time.

In another embodiment, an appropriate amount of a repellent compound orcomposition disclosed herein can be combined with an appropriatepropellant and used as a spray for application into or onto the intendedlocation.

In another embodiment, a repellent compound or composition disclosedherein can be impregnated into a compatible matrix. As used herein, theterm “compatible matrix” refers to any material in which one or morerepellent compounds or compositions disclosed herein are either soluble,miscible, or penetrate into and where the material does notsignificantly alter or degrade a repellent activity of the one or morerepellent compounds/compositions. In aspects of this embodiment, acompatible matrix does not significantly alter or degrade a repellentactivity of one or more repellent compounds/compositions over a periodof, e.g., at least 7 days, at least 14 days, at least 21 days, at least28 days, at least 35 days, at least 42 days, at least 49 days, at least56 days, or at least 63 days. Impregnation of a repellentcompound/composition into the compatible matrix can be achieved by anywell known methods known in the art. For example, a repellent compoundor composition disclosed herein may be dissolved into a compatiblevolatile solvent and the resulting solution added to the matrixwhereupon evaporation of the solvent results in impregnation of therepellent compound into the compatible matrix. In this regard, thematrix can be cotton twine, polymers such as, e.g., polyvinyls,polyisoprenes, polyethylene, polypropylene or copolymers thereof, orpolybutenes. In another example, heating thins a compatible matrix andthen a repellent compound/composition is added directly thereto. Themixture can then be combined with twine or other compatible matrices. Acompatible matrix disclosed herein may be employed by itself orincorporated into a device used to house the matrix.

In another embodiment, a repellent compound or composition disclosedherein can be incorporated into a controlled-release device whichdispenses the repellent compound or composition over time in a regulatedor predictable manner. A controlled-release device disclosed herein maybe employed by itself or incorporated into another device used to housethe controlled-release device.

One type of controlled-release device is a “reservoir” device where therepellent compound or composition disclosed herein forms a coresurrounded by an inert diffusion barrier. An inert diffusion barrierincludes membranes that are non-porous, homogeneous polymeric films,through which transport occurs by a process of dissolution of thepermeating species in the polymer at one interface and diffusion down agradient in thermodynamic activity.

These membranes are usually referred to as solution-diffusion membranes.Another class of inert diffusion barrier includes the porous and/orfibrous barriers such as, for example, hollow fibers, porous and/orfibrous materials, in which a repellent compound or composition diffusesmainly by capillary forces or is introduced into the material byimpregnation. Other less common reservoir devices are designed to enablediffusion to take place by mechanical pumping or under external forces,such as, e.g., gravity, electrical field, vacuum, or centrifugal forces.A reservoir device can exist in a variety of shapes, and can bedegradable or non-degradable.

In an aspect of this embodiment, a reservoir device is a microcapsulecomprising a core of a repellent compound or composition disclosedherein surrounded by a coating or shell of, e.g., a polyvinyl chloride(PVC) polyvinyl acetate (PVA) plastic. Size typically varies from about1 μm to about 1000 μm and can have irregular or geometric shapes. Corepayload usually varies from 0.1 to 98 weight percent. Encapsulationprocesses are often loosely classified as either chemical or mechanical.Examples of chemical processes include but are not limited to complexcoacervation, polymer-polymer incompatibility, interfacialpolymerization in liquid media, in situ polymerization, in-liquiddrying, thermal and ionic gelation in liquid media, desolvation inliquid media, starch-based chemistry processes, trapping incyclodextrins, and formation of liposomes. Examples of mechanicalprocesses include but are not limited to spray drying, spray chilling,fluidized bed, electrostatic deposition, centrifugal extrusion, spinningdisk or rotational suspension separation, annular-jet encapsulation,polymerization at liquid-gas or solid-gas interface, solventevaporation, pressure extrusion or spraying into solvent extractionbath.

Another type of controlled-release device is a “monolithic” device wherea repellent compound or composition disclosed herein is dissolved ordispersed throughout a substantially inert matrix from which therepellent compound or composition disclosed herein is graduallyreleased. Non-limiting examples of matrices included in a monolithicdevice include various gels, waxes, gelatins, natural resins, rubbers,elastomers, synthetic and natural polymers. A monolithic device canexist in a variety of shapes, and can be degradable or non-degradable.Size can vary depending on the application. For example, a monolithicdevice can be produced as a microcapsule having a size of about 1 μm toabout 1000 μm with irregular or geometric shapes. As another example, amonolithic device can have a size of about 1 mm to about 10 cm withirregular or geometric shape.

A controlled-release device disclosed herein can be a liquid compositionor a solid composition. A liquid sustained-release formulation includesa repellent compound or composition disclosed herein, a solvent, andtypically further comprise of surface active agents to render thecomposition readily dispersible in water, such agents include a wettingagent, an emulsifying agent, or a dispersing agent. In one embodiment, aliquid form of a sustained-release formulation is an emulsionformulation, such as, e.g., a water in oil (w/o) emulsion or oil inwater (o/w) emulsion. Non-limiting examples of oils include vegetableoils and mineral oils. Droplet size can vary from the nanometer scale(colloidal dispersion) to several hundred microns. A variety ofsurfactants and thickeners are usually incorporated in the formulationto modify the size of the droplets, stabilize the emulsion, and modifythe release.

A solid form of controlled-release device comprises a solid substratelike porous particulates such as silica, perlite, talc, clay,pyrophyllite, diatomaceous earth, gelatin and gels, polymers (e.g.,polyurea, polyurethane, polyamide, polyester, etc.), polymericparticles, or cellulose. These include, for example, hollow fibers,hollow tubes or tubing which release a repellent compound or compositiondisclosed herein through the walls, capillary tubing which releases thecompound or composition out of an opening in the tubing, polymericblocks of different shapes, e.g., strips, blocks, tablets, discs, whichrelease the compound out of the polymer matrix, membrane systems whichhold the repellent compound within an impermeable container and releaseit through a measured permeable membrane, and combinations of theforegoing. Examples of other dispensing means are polymer laminates,polyvinyl chloride pellets, and microcapillaries.

Controlled release can also be achieved by a number of other methodssuch as, e.g., complexation of a repellent compound or compositiondisclosed herein, slowly dissolving coatings, erosion, microbial action,or use of derivatives or new compounds of reduced solubility orvolatility.

In aspects of this embodiment, a controlled-release device releases arepellent compound or composition disclosed herein with substantiallyzero order release kinetics over a period of, e.g., about 1 day, about 3days, about 7 days, about 15 days, about 30 days, about 45 days, about60 days, about 75 days, or about 90 days. In other aspects of thisembodiment, a controlled-release device releases a repellent compound orcomposition disclosed herein with substantially zero order releasekinetics over a period of, e.g., at least 1 day, at least 3 days, atleast 7 days, at least 15 days, at least 30 days, at least 45 days, atleast 60 days, at least 75 days, or at least 90 days. In other aspectsof this embodiment, a controlled-release device releases a repellentcompound or composition disclosed herein with substantially zero orderrelease kinetics over a period of between, e.g., about 1 day to about 7days, about 1 day to about 15 days, about 1 day to about 30 days, about7 days to about 30 days, about 15 days to about 45 days, about 30 daysto about 60 days, about 45 days to about 75 days, or about 60 days toabout 90 days.

In aspects of this embodiment, a controlled-release device releases arepellent compound or composition disclosed herein with substantiallyfirst order release kinetics over a period of, e.g., about 1 day, about3 days, about 7 days, about 15 days, about 30 days, about 45 days, about60 days, about 75 days, or about 90 days. In other aspects of thisembodiment, a controlled-release device releases a repellent compound orcomposition disclosed herein with substantially first order releasekinetics over a period of, e.g., at least 1 day, at least 3 days, atleast 7 days, at least 15 days, at least 30 days, at least 45 days, atleast 60 days, at least 75 days, or at least 90 days. In other aspectsof this embodiment, a controlled-release device releases a repellentcompound or composition disclosed herein with substantially first orderrelease kinetics over a period of between, e.g., about 1 day to about 7days, about 1 day to about 15 days, about 1 day to about 30 days, about7 days to about 30 days, about 15 days to about 45 days, about 30 daysto about 60 days, about 45 days to about 75 days, or about 60 days toabout 90 days.

Regardless of the method of application, the amount of a repellentcompound or composition disclosed herein is a repellent effectiveamount, i.e., it is an amount sufficient to retard the movement ofmosquitoes or other blood-feeding arthropod to the selected individualor location. In aspects of this embodiment, a repellent compound orcomposition disclosed herein is applied at a rate of, e.g., about 0.01mg/m², about 0.025 mg/m², about 0.05 mg/m² about 0.075 mg/m², about 0.1mg/m², about 0.25 mg/m², about 0.5 mg/m², about 0.75 mg/m² about 1mg/m², about 2.5 mg/m², about 5 mg/m², about 7.5 mg/m², about 10 mg/m²,or about 50 mg/m². In other aspects of this embodiment, a repellentcompound or composition disclosed herein is applied at a rate of, e.g.,at least 0.01 mg/m², at least 0.025 mg/m², at least 0.05 mg/m², at least0.075 mg/m², at least 0.1 mg/m², at least 0.25 mg/m², at least 0.5mg/m², at least 0.75 mg/m², at least 1 mg/m², at least 2.5 mg/m², atleast 5 mg/m², at least 7.5 mg/m² at least 10 mg/m², or at least 50mg/m². In yet other aspects of this embodiment, a repellent compound orcomposition disclosed herein is applied at a rate of, between e.g.,about 0.01 mg/m² to about 50 mg/m², about 0.01 mg/m² to about 10 mg/m²,about 0.01 mg/m² to about 1 mg/m², about 0.01 mg/m² to about 0.1 mg/m²,about 0.05 mg/m² to about 50 mg/m², about 0.05 mg/m² to about 10 mg/m²,about 0.05 mg/m² to about 1 mg/m², about 0.05 mg/m² to about 0.1 mg/m²,about 0.05 mg/m² to about 5 mg/m², or about 0.05 mg/m² to about 0.5mg/m².

Aspects of the present specification may also be described as follows:

1. A composition comprising a plurality of repellent compounds fromdiverse sources having mosquito repellent activity.2. A composition comprising a combination of two or more compoundshaving insect and/or arachnid repellent activity, with the said two ormore compounds being selected, independently, from carvacrol compounds,cumin compounds, and cinnamate compounds.3. The composition according to embodiment 1 or 2, wherein the pluralityof repellent compounds includes a carvacrol compound, a cumin compound,a cinnamate compound, or any combination thereof.4. A composition comprising a carvacrol compound and one or moreadditional repellent compounds having mosquito and blood-suckingarthropod repellent activity.5. The composition according to any one of the preceding embodiments,wherein one or more additional repellent compounds includes a cumincompound, a cinnamate compound, or any combination thereof.6. A composition comprising a carvacrol compound and a cumin compound,wherein the composition has mosquito repellent activity.7. A composition comprising a carvacrol compound and a cinnamatecompound, wherein the composition has mosquito and blood-suckingarthropod repellent activity.8. A composition comprising a carvacrol compound, a cumin compound, anda cinnamate compound, herein the composition has mosquito andblood-sucking arthropod repellent activity.9. The composition according to embodiments 1-8, wherein the compositionhas mosquito and blood-sucking arthropod repellent activity.10. The composition according to embodiment 9, wherein presence of thecomposition repels mosquitoes and blood-sucking arthropods by at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95%, as compared to not having thecomposition present.11. The composition according to embodiments 1-10, wherein thecomposition reduces an interaction between a blood-feeding arthropod anda mammal, in particular a mosquito-mammalian host interaction.12. The composition according to embodiment 11, wherein the compositionreduces said interaction by, e.g., at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95%.13. The composition according to embodiments 1-12, wherein thecomposition reduces an ability of a blood-feeding arthropod, inparticular a mosquito, to obtain a blood-meal from a mammal.14. The composition according to embodiment 13, wherein the compositionreduces said ability by, e.g., at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%.15. The composition according to embodiments 1-14, wherein the appliedcomposition comprises about 0.01 μg/cm² to about 50 μg/cm² of acarvacrol compound, about 0.1 μg/cm² to about 10 μg/cm² of a carvacrolcompound, or about 0.5 μg/cm² to about 5 μg/cm² of a carvacrol compound.16. The composition according to embodiments 1-15, wherein the appliedcomposition comprises about 0.01 μg/cm² to about 50 μg/cm² of a cumincompound, about 0.1 μg/cm² to about 10 μg/cm² of a cumin compound, orabout 0.5 μg/cm² to about 5 μg/cm² of a cumin compound.17. The composition according to embodiments 1-16, wherein the appliedcomposition comprises about 0.01 μg/cm² to about 50 μg/cm² of acinnamate compound, about 0.1 μg/cm² to about 10 μg/cm² of a cinnamatecompound, or about 0.5 μg/cm² to about 5 μg/cm² of a cinnamate compound.18. The composition according to embodiments 1-17, wherein the appliedcomposition comprises about 0.01 nmole/cm² to about 200 nmole/cm² of acarvacrol compound, about 0.1 nmole/cm² to about 50 nmole/cm² of acarvacrol compound, about 1 nmole/cm² to about 30 nmole/cm² of acarvacrol compound, or about 2.5 nmole/cm² to about 25 nmole/cm² of acarvacrol compound.19. The composition according to embodiments 1-18, wherein the appliedcomposition comprises about 0.01 nmole/cm² to about 200 nmole/cm² of acumin compound, about 0.1 nmole/cm² to about 50 nmole/cm² of a cumincompound, about 1 nmole/cm² to about 30 nmole/cm² of a cumin compound,or about 2.5 nmole/cm² to about 25 nmole/cm² of a cumin compound.20. The composition according to embodiments 1-19 wherein the appliedcomposition comprises about 0.01 nmole/cm² to about 200 nmole/cm² of acinnamate compound, about 0.1 nmole/cm² to about 50 nmole/cm² of acinnamate compound, about 1 nmole/cm² to about 30 nmole/cm² of acinnamate compound, or about 2.5 nmole/cm² to about 25 nmole/cm² of acinnamate compound.21. The composition according to embodiments 1-20, wherein the appliedcomposition comprises about 0.01 μg/cm² to about 50 μg/cm² of acarvacrol compound, about 0.01 μg/cm² to about 50 μg/cm² of a cumincompound disclosed herein, and about 0.01 μg/cm² to about 50 μg/cm² of acinnamate compound.22. The composition according to embodiments 1-21, wherein the appliedcomposition comprises about 0.1 μg/cm² to about 10 μg/cm² of a carvacrolcompound, about 0.1 μg/cm² to about 10 μg/cm² of a cumin compound, andabout 0.1 μg/cm² to about 10 μg/cm² of a cinnamate compound.23. The composition according to embodiments 1-22, wherein the appliedcomposition comprises about 0.5 μg/cm² to about 5 μg/cm² of a carvacrolcompound, about 0.5 μg/cm² to about 5 μg/cm² of a cumin compound, andabout 0.5 μg/cm² to about 5 μg/cm² of a cinnamate compound.24. The composition according to embodiments 1-23, wherein the appliedcomposition comprises about 0.01 nmole/cm² to about 100 nmole/cm² of acarvacrol compound, about 0.01 nmole/cm² to about 200 nmole/cm² of acumin compound, and about 0.01 nmole/cm² to about 200 nmole/cm² of acinnamate compound.25. The composition according to embodiments 1-24, wherein the appliedcomposition comprises about 0.1 nmole/cm² to about 50 nmole/cm² of acarvacrol compound, about 0.1 nmole/cm² to about 50 nmole/cm² of a cumincompound, and about 0.1 nmole/cm² to about 50 nmole/cm² of a cinnamatecompound.26. The composition according to embodiments 1-25, wherein the appliedcomposition comprises about 1 nmole/cm² to about 30 nmole/cm² of acarvacrol compound, about 1 nmole/cm² to about 30 nmole/cm² of a cumincompound, and about 1 nmole/cm² to about 30 nmole/cm² of a cinnamatecompound.27. The composition according to embodiments 1-26, wherein the appliedcomposition comprises about 2.5 nmole/cm² to about 25 nmole/cm² of acarvacrol compound, about 2.5 nmole/cm² to about 25 nmole/cm² of a cumincompound, and about 2.5 nmole/cm² to about 25 nmole/cm² of a cinnamatecompound.28. The composition according to embodiments 1-28, wherein the carvacrolcompound includes carvacrol, thymol, or any combination thereof.29. The composition according to embodiments 1-28, wherein the cumincompound includes cumin alcohol, cumin aldehyde, cuminic acid, or anycombination thereof.30. The composition according to embodiments 1-29, wherein the cinnamatecompound includes cinnamate, methyl cinnamate, ethyl cinnamate, butylcinnamate, isobutyl-cinnamate, N-butyl-cinnamate, isopropyl-cinnamate,E-cinnamyl acetate, cinnamaldehyde, E-cinnamaldehyde, Z-cinnamaldehyde,o-methoxycinnamaldehyde, or any combination thereof.31. A method of reducing bites by blood-feeding arthropods, inparticular insects and ticks in an individual, the method comprising thestep of applying a composition according to embodiments 1-30 to theindividual, wherein application of the composition repels a mosquito,sand fly (or an insect) and other blood-sucking arthropods from theindividual, thereby reducing bites for a blood-meal.32. The method according to embodiment 31, wherein application of thecomposition is by topical administration.33. A method of reducing a mosquito infestation to a location, themethod comprising the steps of applying a composition according toembodiments 1-30 to the location, wherein the application repels insectsand/or arachnids, in particular mosquitoes, sand flies and/or ticks fromthe location, thereby reducing the infestation.34. The method according to embodiment 33, wherein the location is aplant or group of plants, a particular area of land, or a man-madestructure.35. The method according to embodiment 33, wherein the man-madestructure is a commercial building, a house, a shed, a livestockmaintenance area, other physical structure, or any part thereof.36. A compound or composition having a repellency activity with respectto blood-sucking arthropods, in particular insect and/or arachnidrepellent activity, preferably mosquito repellency activity, wherein thecompound binds to Anopheles gambiae OBP1, OBP3, OBP4, OBP5, OBP20,OBP47, and/or or one or more different mosquito OBPs.37. The compound or composition according to embodiment 35, whereinpresence of the composition repels blood-sucking arthropods, inparticular insect and/or arachnids, for example mosquitoes by at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95%, as compared to not having thecomposition present.38. The compound or composition according to embodiment 35 or 36,wherein the composition reduces an interaction between blood-feedingarthropod, such as a blood-feeding insect and/or arachnid, and a mammalor bird, in particular a mosquito-mammalian host interaction.39. The compound or composition according to embodiment 37, wherein thecomposition reduces interaction of said blood-feeding arthropod with amammal or bird by, e.g., at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%.40. The compound or composition according to embodiments 35-38, whereinthe composition reduces an ability of a blood-feeding arthropod, inparticular an insect and/or arachnid, such as a mosquito to obtain ablood meal from a mammal or a bird.41. The compound or composition according to embodiment 39, wherein thecomposition reduces an ability of said blood-feeding arthropod, inparticular said mosquito to obtain a blood-meal from a mammal or birdby, e.g., at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, or at least 95%.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification, including those pertaining to the compounds,compositions, traps, methods or uses of repellents for mosquitoes andother blood-sucking arthropods.

Example 1 Purification of Essential Oils from Plant Material

This example illustrates how to extract an essential oil from plantmaterial.

To extract an essential oil from a plant, material was collected frommostly aromatic plant species. Essential oils were obtained bysteam-distilling 50-70 g of plant samples (examples are shown inTable 1) for 4 hours in a modified Clevenger distillation apparatusequipped with a water-cooled oil receiver to reduce hydrodistillationartifacts. The volatiles, which were carried by water vapor, werecondensed and trapped in a layer of diethyl ether. The ether layer wasdried over magnesium sulfate to remove residual water, concentratedunder a gentle stream of nitrogen and each essential oil was stored at−20° C.

TABLE 1 Plant Species and Parts Used to Extract Essential Oils (EOs) EOSpecies No. Code Species Part used 1 Lam27 Salvia sclarea flowers 3Lam12 Lavandula stoechas aerial plant 5 Lam22.1 Coridothymus capitatusaerial plant 6 Lam05.4 Origanum majorana aerial plant 9 Lam23 Origanumheracleoticum aerial plant 11 Lam09.1 Salvia fruticosa aerial plant 14Lam07.2 Origanum vulgare flowers 16 Lau02 Cinamomum camphora leaves 20Ana02 Schinus cf. molle leaves 21 Com01 Santolina chamaecyparissusaerial plant 29 Gut02 Hypericum sp. leaves 36 Gut01 Hypericum balearicumaerial plant 41 Cup04 Juniperus foenicae shoots 46 Lam02 Rosmarinusofficinalis aerial plant 47 Lam28 Ocimum basilicum aerial plant 54 Myr02Eucalyptus camaldulensis leaves 55 Pin03 Pinus halepensis shoots 166Rut08 Citrus sinensis fruit peel 169 Lam07 Origanum sp. aerial plant 171Lam01 Satureja thymbra aerial plant 174 Car02 Dianthus caryophyllus dryseeds 176 Ill01 Illicium verum dry fruits 180 Umb08 Cuminum cyminum dryseeds 181 Umb09 Pimpinella anisum dry seeds 189 Cup05 Juniperus sp. dryfruits 190 Umb10 Carum carvi dry seeds

Example 2 Screening of Essential Oils Using a OBP Binding CompetitionAssay

To identify a mosquito repellent disclosed herein, essential oils werefirst screened for candidate compounds based upon the ability of thatcompound to bind mosquito OBPs using a fluorescent binding competitionassay. The fluorescence binding competition assay employed in theidentification of candidate compounds was based on displacement of thefluorescent probe N-phenyl-1-naphthylamine (1-NPN) from an OBP byvarious compounds present in the purified essential oils.

To construct an expression construct comprising an OBP, a full-lengthcDNA for an OBP was subcloned into a pIE1/153A expression vector[Farrell et al (1998). High-level expression of secreted glycoproteinsin transformed lepidopteran insect cells using a novel expressionvector. Biotechnol Bioeng 60(6): 656-663; Lu et al (1997). A baculovirus(Bombyx mori nuclear polyhedrosis virus) repeat element functions as apowerful constitutive enhancer in transfected insect cells. J Biol Chem272(49): 30724-30728] in a manner that enabled C-terminal tagging of theexpressed OBPs with a 6xHis purification and a c-Myc epitope tag aspreviously described [Douris et al (2006). Stably transformed insectcell lines: tools for expression of secreted and membrane-anchoredproteins and high-throughput screening platforms for drug andinsecticide discovery. Adv Virus Res 68: 113-156]. pIE1/153A expressionconstructs (pIE1/153A.OBPx) were made using the following polynucleotidesequences: OBP1 (SEQ ID NO: 1), OBP3 (SEQ ID NO: 2), OBP4 (SEQ ID NO:3), OBP5 (SEQ ID NO: 4), OBP20 (SEQ ID NO: 5) and OBP47 (SEQ ID NO: 6).These six OBPs were selected since each is highly expressed in theantennae of A. gambiae with a strong female bias [Biessmann et al(2002). Isolation of cDNA clones encoding putative odorant bindingproteins from the antennae of the malaria-transmitting mosquito,Anopheles gambiae. Insect Mol Biol 11(2): 123-132].

To generate a transiently transformed cell line, weekly subculturedHIGHFIVE™ cells (Invitrogen, Inc., Carlsbad, Calif.), grown at 28° C. ineither IPL-41 insect cell culture medium (Genaxxon Biosciences)supplemented with 10% fetal bovine serum (Life Technologies, Inc.,Carlsbad, Calif.) or serum free ESF 921 medium (Expression systems LLC),were transfected with a pIE1/153A expression construct using Lipofectin(Invitrogen, Inc., Carlsbad, Calif.) or Escort IV reagents (Sigma, St.Louis, Mo.) according to the manufacturers' instructions. To generate astable transformed cell line, HIGHFIVE™ cells were co-transfected with apIE1/153A expression construct and pEApac, [Douris et al (2006). Stablytransformed insect cell lines: tools for expression of secreted andmembrane-anchored proteins and high-throughput screening platforms fordrug and insecticide discovery Adv Virus Res 68: 113-156], a plasmidconferring resistance to puromycin, at a molar ratio of 100pIE1/153A.OBPx to 1 pEApac. Transformed cell lines were selected withina period of 2-3 weeks and maintained in the appropriate growth medium inthe presence of 50 μg/mL gentamycin (Invitrogen, Inc., Carlsbad, Calif.)and 15 or 50 μg/ml puromycin (Sigma, St. Louis, Mo.).

To express and purify an OBP, 500 mL cultures of transformed cells weregrown in a BIOWAVE® 2SPS bioreactor (Wave Biotech AG, Switzerland) for7-8 days to a cell density of about 2×10⁶ cells/mL in serum containingor about 7×10⁶ cells/mL in serum free medium. Culture supernatants werecollected, pH adjusted to 8.0 using 0.5 M sodium phosphate buffer (pH8.0) and batch bound overnight to Ni-NTA agarose resin (Qiagen Inc.,Valencia, Calif.). The resin was poured into a column and then washedonce with with 200 mL of 10 mM imidazole in 50 mM phosphate buffer (pH8.0) containing 300 mM NaCl and then washed once with 100 mL of 20 mMimidazole in 50 mM phosphate buffer (pH 8.0) containing 300 mM NaCl.Protein was eluted with 250 mM imidazole in 50 mM phosphate buffer (pH8.0) containing 300 mM NaCl. Protein samples from all fractions wereanalyzed on 15% sodium dodecyl sulphate (SDS)-polyacrylamide gels,stained with silver nitrate and also electroblotted on nitrocellulosemembranes for visualization by Western Blot analysis using theantibodies against the c-Myc tag (Santa Cruz, Biotechnology Inc., SantaCruz, Calif.) and ECL detection (Amersham Pharmacia Biotech). OBPcontaining fractions were concentrated and loaded onto a Superdex75 gelfiltration column (GE Healthcare) equilibrated with 10 mM Tris-HCl pH8.0 containing 200 mM NaCl, and the corresponding peak of protein elutedwas identified by SDS-PAGE analysis. Fractions were subsequentlysubjected to buffer exchange in 10 mM Tris-HCl pH 8.0, 50 mM NaCl, usingAmicon Ultra-15 Centrifugal filter Devices (Millipore).

To conduct a fluorescence binding competition assay, the concentrationof each purified OBP required for maximal binding of 1-NPN was firstdetermined in binding assays using increasing OBP amounts. All testedOBPs bound 1-NPN at about 1:1 molar ratios. Essential oils were thentested for binding using the minimum amount of each purified OBP thatyielded maximal binding of the fluorescent probe. Each essential oil wasadded at a defined dilution ranging from 1/12,500 to 1/100,000. Theprobe was excited at 337 nm and emission spectra were recorded between386-460 nm (peak emission in the presence of an OBP was between 402-406nm). Emission spectra were recorded on an Infinite M-200 fluorimeter(Tecan Trading AG, Switzerland). For Kd value calculations, thedissociation constant of 1-NPN (Kp) was determined by fluorescencemeasurements of solutions containing OBP and 1-NPN. Bound candidatecompound concentration was calculated from the fluorescence intensitiesassuming 100% functional protein and an 1:1 [OBP]:[1-NPN] stoichiometryat saturation. The Kd, was calculated using a non-linear-regression dataanalysis program (GraFit).

The results shown in Table 2 indicate that of 26 essential oilsexamined, 11 contained at least one compound which competed with 1-NPNfor binding to one or more of the tested OBPs, while the remaining 15essential oils appeared not to have any compounds that could displace1-NPN from any of the tested OBPs (Table 2). For the essential oilslisted in table 2, when an oil contained one or more compounds thatbound to several OBPs, displacement of 1-NPN ranged from 11% to 46%. Thebinding experiments also identified OBP4 as the protein with the mostpromiscuous binding behavior (Table 2).

TABLE 2 Binding Activity of Essential Oils (EOs) and Their Fractions (a,b, c) to OBPs Species Max % OBP EO No Code OBP1 OBP3 OBP4 OBP5 OBP20OBP47 binding  1 Lam27 — — — — — —  0%  3 Lam12 — — — — — —  0%  5Lam22.1 — ✓ ✓ — ✓ — 21%  6 Lam05.4 — — — — ✓ — 17%  9 Lam23 — — ✓ — ✓ —19%  9b — ND ND — ✓ ✓ 22%  9c — ND ND — ✓ ✓ 26% 11 Lam09.1 — — — — — — 0% 14 Lam7.2 ✓ — ✓ — ✓ — 28% 16 Lau02 — — — — — —  0% 20 Ana02 — — — —— —  0% 21 Com01 ✓ ✓ ✓ ✓ ✓ ✓ 25%  21b ✓ ✓ ✓ ✓ ✓ ✓ 43%  21c ✓ ✓ ✓ ✓ ✓ ✓39% 29 Gut02 ✓ ✓ — ✓ — ✓ 12%  29b ✓ — ✓ ✓ — ND 23%  29c ✓ ✓ ✓ ✓ ✓ ND 24%36 Gut01 — — — — — —  0% 41 Cup04 — — — — — —  0% 46 Lam02 — — — — — — 9% 47 Lam28 ✓ ✓ ✓ ✓ ✓ ✓ 46%  47a ✓ ✓ ✓ ✓ ✓ ND 39%  47b ✓ ✓ ✓ ✓ ✓ ND 60% 47c — — ✓ ✓ — ND 21% 54 Myr02 ✓ ✓ — ✓ — — 11% 55 Pin03 — — — — — —  5%166  Rut08 — — — — — —  0% 169  Lam07 — — ✓ — — — 35% 171  Lam01 — — — —— —  0% 174  Car02 — — — — — —  0% 176  III01 — — — — — —  0% 180  Umb08✓ ✓ — ✓ ✓ ✓ 21% 180a — — ✓ ✓ — — 13% 180b ✓ ✓ ✓ ✓ ✓ ✓ 44% 180c ✓ ✓ ✓ ✓ ✓✓ 81% 181  Umb09 — — — — — —  0% 189  Cup05 — — — — — —  0% 190  Umb10 ✓— ✓ ✓ ✓ ✓ 34% 190b ✓ ✓ ✓ ✓ ✓ ND 17% 190c ✓ — ✓ ✓ ✓ ND 24% ✓, bindingcompetition (>9%); —, no binding competition; ND, not done; a, b, cdesignations, see example 4 description.

Example 3 Testing of Essential Oils Containing OBP Binding CompoundsUsing a Repellence Assay

To test the behavior of female mosquitoes to an essential oil containingOBP binding components, a previously described repellence assay [Kroberet al. (2010). An In Vitro Assay for Testing Mosquito RepellentsEmploying a Warm Body and Carbon Dioxide as a Behavioral Activator.Journal of the American Mosquito Control Association 26(4) 381-386] wasconducted that measures the number of mosquito landings on a warmsurface onto which an essential oil containing at least one candidatecompound was applied.

To conduct a repellency assay, a selected essential oil or ethanolnegative control was applied to a warm body at a dose of about 3.8μg/cm² and allowed to dry for 40 seconds in order to allow evaporationof the solvent before introduction into the test cage with mosquitoes.As positive control for repellence, DEET dissolved in ethanol wasapplied to the warm body at the same dose. The treated warm body inconjunction with a pulse of carbon dioxide was introduced into the testcage containing fifty Anopheles gambiae (Giles) ss strain 16CSS femalemosquitoes that were 4-11 days old and the number of mosquito landingson the warm body occurring within a 2-minute period was counted. Eachcandidate compound was tested in five different cages (i.e., 250 femalemosquitoes) in a randomized design, and the ethanol control and up tofour candidate compounds were tested in each cage. Raw data on numbersof landings by mosquitoes were analyzed using a linear mixed modelaccounting for treatment, experimental day, the individual cage, thenumber of females per cage and the level of the carbon dioxide pulse inppm using R (R Development Core Team, 2010) and the packages LME4 (Batesand Maechler, Ime4: Linear mixed-effects models using {S4} classes.{R}package version 0.999375-32, 2009) and NLME (Pinheiro et al, nlme:Linear and Nonlinear Mixed Effects Models, 2010; the R Development CoreTeam, R: A Language and Environment for Statistical Computing, 2010).Repellent values were calculated from counted landings on treatmentsversus the overall landings on pooled controls.

The results of the tests on 26 of the essential oils shown in Table 1indicate that of the 11 essential oils competing with 1-NPN for bindingto one or more OBPs (Table 2), 5 displayed very strong repellentactivities in the warm body assay at a dose of 3.8 mg/cm² (Table 3).Essential oils extracted from Coridothymus capitatus, Origanum majorana,Origanum heracleorticum, Origanum vulgare and Origanum sp. exhibitedrepellency well above 90%, similar to the activity displayed by DEET, aknown repellent, at the same dose (Table 3). The results also revealedthat an additional essential oil, that of Santolina chamaecyparissus,displayed lower but still moderate repellent activity (median repellenceof 72%). Warm body tests with the 15 essential oils that failed to bindto any of the six OBPs tested to any measurable degree showed that onlyone “OBP binding-negative” essential oil, that of Dianthus caryophyllus,had moderate repellent activity (median repellence of 74%).

TABLE 3 Repellence Activity of Essential Oils (EO) and their Fractions(a, b, c) Rep Median P value P value EO Fraction Species % control DEET1 Salvia sclarea 33 0 0 3 Lavandula stoechas 48 0 0 5 Coridothymuscapitatus 97 0 0.89 6 Origanum majorana 95 0 0.88 9 Origanumheracleoticum 93 0 0.49  9b 101 0 0.77  9c 95 0 0.72 11 Salvia fruticosa23 0.01 0 14 Origanum vulgare 95 0 0.82 16 Cinamomum camphora 12 0.12 020 Schinus cf. molle 29 0 0 21 Santolina 72 0 0.01 chamaecyparissus 21b36 0 0 21c 9 0.24 0 29 Hypericum sp. 38 0 0 29b 48 0 0 29c 83 0 0.17 36Hypericum balearicum 34 0 0 41 Juniperus foenicae 28 0 0 46 Rosmarinusofficinalis −7 0.54 0 47 Ocimum basilicum 14 0.01 0 47a 49 0 0 47b 24 00 47c 30 0.03 0 54 Eucalyptus 22 0 0 camaldulensis 55 Pinus halepensis32 0 0 166 Citrus sinensis 28 0 0 169 Origanum sp. 93 0 0.66 171Satureja thymbra 6 0.50 0 174 Dianthus caryophyllus 74 0 0.01 176Illicium verum 26 0 0 180 Cuminum cyminum 7 0.39 0 180a  4 0.62 0 180b 25 0 0 180c  66 0 0 181 Pimpinella anisum 41 0 0 189 Juniperus sp. 37 00 190 Carum carvi 45 0 0 190b  29 0 0 190c  64 0 0

Example 4 Screening of Essential Oil Fractions Using an OBP BindingCompetition and Repellency Assay

Since the tested essential oils are complex mixtures of differentchemical compounds, both binding activity data and behavioral effectsmay be affected by low concentrations of active compounds in eachessential oil, possible functional interference interactions betweencompounds, or a combination of both effects. As such, both bindingactivity and behavioral assays were repeated on fractions of selectedessential oils.

Essential oils purified in Example 1 were further fractionated usingsolid phase extraction (SPE) on SEP-PAK® Plus Alumina A (Waters) andeluting at a velocity of 1 drop/second with the following solvents insequence: Fraction A, 3 mL pentane; Fraction B 6 mL pentane/diethylether 90:10; and Fraction C, 3 mL diethyl ether. Each fraction wasconcentrated under a gentle stream of nitrogen and stored at −20° C.until use. The fluorescence binding competition assay was performed asdescribed in Example 2.

Ten essential oils were separated into three fractions and each fractiontested in the fluorescence binding competition assay as described inExample 2. The results indicate that fractions from at least seven ofthe essential oils examined appeared enriched for at least one compoundwhich competed with 1-NPN for binding to one or more of the tested OBPs(Table 2). Of those oil fractions that had a compound that could competewith 1-NPN, the percent displacement ranged form about 13% to about 81%.

Fractions from 6 selected essential oils were also tested using therepellence assay as described in Example 3. The analysis of thesefractions revealed that fractionation resulted in enrichment ofbioactive components or removal of competing inhibitory bioactivitiesfrom the fractions displaying enhanced repellent activities or both. Forexample, essential oil of Cuminum cyminum, which was inactive in thewarm body assay (median repellency of 7%), showed that fraction 108C wasfound to possess significant repellent activity (median repellency of66%; Table 3). As another example, essential oil of Santolinachamaecyparissus was found to be more active than any of the fractionsderived from it (Table 3), a finding suggesting possible combinatorialeffects amongst two or more bioactive components partitioning in thedifferent fractions.

Example 5 Candidate Compound Identification Using GasChromatography-Linked Electroantennogram Recordings and MassSpectrometry

In order to identify compounds present in essential oils with mosquitorepellent activity, selected essential oils exhibiting affinity to OBPsand repellent activity in mosquitoes were subjected to gaschromatography-coupled electroantennogram (GC-EAG) analysis (FIG. 1) andindividual compounds eliciting strong EAG responses were analyzed by gaschromatography-coupled mass spectrometry (GC-MS).

Electrophysiological recordings from whole mosquito antenna: CouplingEAG recordings from an A. gambiae antenna to the effluent of ahigh-resolution gas chromatographic (GC) column permitted thedetermination of the elution profiles of biologically active componentsof essential oils showing repellence. Recordings were made from to 4-9day-old female mosquitoes. The head was excised at the occipital openingand placed on the reference glass electrode containing Hayes mosquitoRinger [Hayes (1953). Determination of a physiological saline for Aedesaegypti (L.). J Econ Entomol 46: 624-627] mounted in a humidifiedair-stream (90-98% RH; Guerin and Visser (1980). ElectroantennogramResponses of the Carrot Fly, Psila rosae, to Volatile Plant Components.Physiological Entomology 5(2): 111-119] and its antenna exposed tocompounds eluting from the GC column (Biessamann et al. (2010). TheAnopheles gambiae odorant binding protein 1 (AgamOBP1) mediates indolerecognition in the antennae of female mosquitoes. PLoS One 5(3): e9471].The EAG response was recorded via a glass electrode filled withKaissling sensillum lymph Ringer [Kaissling (1995). Single unit andelectroantennogram recordings in insect olfactory organs. InExperimental Cell Biology of Taste and Olfaction. Current Techniques andProtocols, Spielman AlaB, J. G. (ed), 361-386. Boca Raton, Fla.: CRCPress] brought into contact with the terminal antennal segment whose tipwas cut off. Only antennae showing an EAG response at least double thenoise level to a 1 ml puff of air over 1 μg geranylacetone in a 5 mlstimulus syringe were used for recording EAG responses to the essentialoils.

To conduct a GC-EAG analysis, about 2 μg of an essential oil wasinjected in 1-3 μL of dichloromethane (DCM) onto an apolar capillarycolumn (95% dimethyl polysiloxan with 5% diphenyl polysiloxan, 15 mlong, 0.25 mm i.d., 0.10 μm film thickness; BGB Analytik, Switzerland)installed in a 5300 Carlo Erba Instruments chromatograph. Hydrogen wasused as carrier gas with the oven held at 40° C. for 3 minutes thenheated up 25° C./min to 230° C. and held at the final temperature for 15min. The column effluent was split (50:50) between the flame ionizationdetector (FID) of the chromatograph and the mosquito antennalpreparation. The EAG signal was fed into an AC/DC amplifier (×100) via ahigh impedance preamplifier (×10), recorded on the hard disk of a PC viaa 16-bit analogue-digital IDAC4 interface (Syntech, Netherlands) andmonitored simultaneously with an oscilloscope (Tektronix 5103, USA).Kovats retention indices (KIs) were calculated for the EAG-activecompounds present in the extracts and fractions. FIG. 1 illustrates theresults obtained using the GC-EAG analysis.

This analysis resulted in the identification of a number of compoundsthat were capable of triggering EAG responses in female A. gambiaeantennae. These included carvacrol, β-caryophyllene,(E)-methyl-cinnamate, cumin alcohol, cumin aldehyde, cuminic acid,p-cymene, linalool, α-pinene, β-pinene and safranal, and γ-terpineol.

To conduct a GC-MS analysis, 1 μL of an essential oil extract orfraction thereof was dissolved in 1 mL of DCM and 1 μL of this solutionwas analyzed on an Agilent 7890A GC apparatus coupled with a massselective detector (5975E MSD) and equipped with a Gerstel MPS2XLautosampler. The GC was equipped with a Zebron ZB-5 capillary column (30m×0.25 mm i.d. coated with 5% diphenyl-95% dimethylpolysiloxane, 0.10 μmthickness). The injection port and transfer line were set at 250° C.with helium as carrier gas (at 9.59 psi). Oven temperature was firstincreased from 40° C. to 150° C. at 5° C./min, then to 220° C. at 10°C./min and then to 310° C./min at 30° C./min and held for 8 minutes.Mass spectra were acquired in EI mode (at 70 eV). A set of C10-C24alkanes was injected under the same experimental conditions and KIs werecalculated for the compounds present in the plant extracts. Both therecorded mass spectra and the KIs were compared with those reported inelectronic libraries Nist 0.5, Wiley7, and Adams. Elution profiles werecompared to those obtained from the GC-EAG recordings (above), and KIsof single compounds were compared taking into account the slightdifferences observed due to the use of different carrier gases, so as toidentify compounds eliciting EAG responses from the mosquito antennae.

Example 6 Testing of Candidate Compounds Using a Repellence Assay

To test the behavior of female mosquitoes to a candidate compoundidentified in Example 5, behavioral assays were made using theidentified compounds. The repellent assay was performed as described inExample 3, except that candidate compounds were dissolved in ethanol andtested initially at doses between 1.0 and 5.0 μg/cm² (about 5-31.5nmole/cm²) and, whenever necessary, at lower and/or higher doses aswell. Besides the compounds identified in Example 5, compounds relatedto these were also tested including ethyl-cinnamate and butyl-cinnamate(Table 4).

TABLE 4 Repellence Activity of Compounds Identified in Essential Oilsand Related Compounds Compounds identified in Essential Oils (EOs)Median P value P value Essential nmole per Repellency compared tocompared to CAS # Full name Oil/Fraction cm² (%) ethanol control DEET*134-62-3 DEET 20 98.5 0 ref 499-75-2 carvacrol 169, 5, 14, 169 25 93.3 00.509 1754-62-7  methyl-trans- 47, 47a, 47b 20 14.0 0.001 0 cinnamate536-60-7 cumin alcohol 180, 180c 20 92.3 0 0.121 122-03-2 cumin aldehyde180,180a, 20 13.5 0.0016 0 180b,180c 536-66-3 cuminic acid 180, 180b, 2074.2 0 0.706 180c  89-83-8 thymol 5, 169 31.5 89.7 0 0.203 RelatedCompounds Median P value P value Related nmole per Repellency comparedto compared CAS # Full name compound cm² (%) ethanol control to DEET*103-36-6 (E)-ethyl- (E)-methyl- 20 82.8 0 0.4238 cinnamate cinnamate538-65-8 butyl-cinnamate (E)-methyl- 20 88.7 0 0.1461 cinnamate*Statistically significant differences are those with P values < 0.05

The results, which are summarized in Table 4, show that carvacrol,thymol, cumin alcohol and cuminic acid are repellents with activitycomparable to DEET. Compounds such as (+) and (−) carvone,β-caryophyllene, E-methyl-cinnamate and cuminaldehyde, (+) and (−)limonene, α- and β-pinene, γ-terpinene and safranal displayed only weakor no repellent activity at all in the warm body assay. Interestingly,linalool and p-cymene exhibited properties suggesting attractantactivity consistent with the behavioral effects exerted by fractionsrelative to the parent essential oils.

Compounds related to (E)-methyl-cinnamate, which were also assessed foractivity in the warm body assays due to their structural similarity withthe identified cinnamate, proved to be much stronger repellents thanmethyl-cinnamate. Thus, in contrast to the methyl cinnamate, whichdisplayed only marginal repellent activity (median repellency of 14%;Table 4), ethyl-cinnamate, and butyl-cinnamate were found to have, atcomparable doses, repellencies ranging from 82.8% and 88.7%,respectively, indicative of an effect of the extended hydrophobic chain(Table 4).

Example 7 Testing of Repellent Compound Combinations Using a RepellenceAssay

To test the behavior of female mosquitoes to various combinations ofcandidate compounds identified in Example 6, repellence assays were madeas in Example 3. These repellent assays were made to compare theeffectiveness of the strongest repellent compounds identified in Example6, including, without limitation, carvacrol, ethyl cinnamate, butylcinnamate, and cumin alcohol with that of DEET.

The results indicate that all compounds tested on their own showed astrong increase in repellent activity by increasing the dose from 2 to20 nmol/cm² (Table 5 and FIG. 2). In addition, individually, allcompounds showed strong repellent activity at 20 nmole/cm², althoughDEET was somewhat more repellent. In addition, carvacrol retainedrepellent activity at a concentration as low as 2 nmole/cm². Theseresults also showed that all mixtures containing cavacrol showed astrong increase in repellence with increasing dose (Table 5 and FIG. 2).Furthermore, equimolar binary mixtures of carvacrol with each of theother two compounds at a total concentration of 2.0 nmole/cm² weresignificantly more effective than any of the individual compounds alone,including DEET, at the same final concentration, (P≦0.05). In addition,a binary mixture of butyl cinnamate and cumin alcohol also showed anincrease in repellence with increasing dose (Table 5).

TABLE 5 Dose-Response Repellence of Anopheles gambiae to SelectedCompounds and Their Mixtures Compared to DEET Median repellence %Compound or 2 4 6 10 20 25 compound mixture nmol nmol nmol nmol nmolnmol DEET 2 41 80 96 98 100 Carvacrol 13 55 40 59 —  93 Cumin alcohol −438 46 53 93 — (E)-ethyl-cinnamate −18 18 35 — 83 — Butyl-cinnamate −1 2861 68 89 — Carvacrol + Cumin 28 — 64 81 91 — alcohol Carvacrol + Butyl-55 — 66 84 94 — cinnamate Cumin alcohol + 7 25 — 74 94 — Butyl-cinnamate

FIG. 2. graphically shows repellence indices (predicted values fromgeneralized linear mixed model) of binary mixtures of carvacrol (Carv)(A), cuminic alcohol (CuAlc) (A+B) and butyl cinnamate (CinBut) (A+D) atdifferent doses on the warm body for Anopheles gambiae females ascompared to different doses of carvacrol (A), cumin alcohol (B), ethylcinnamate (CinEt) (C), butyl cinnamate (D) and DEET presented singly.The doses tested were 2, 4, 6 10 and 20 nmol/cm² for the singlecompounds. The binary 50:50 mixtures were tested at 2, 6, 10 and 20nmol/cm², with each compound making up half the amount tested, so that,for example, the 2 nmol/cm² concentration in column A+B represents 1nmol/cm² of A and 1 nmol/cm² of B in a binary mixture. DEET was alsotested at 25 nmol (top response) and carvacrol was tested at theadditional dose of 2.5 nmol (second from left within its column). As anindication, DEET at 20 nmol/cm²=3.8 μg/cm². Mosquitoes were activatedusing a puff of CO₂ from a gas tank upon introduction of the warm bodyinto the test cage containing 50±5 A. gambiae females and mosquitolandings were counted for 2 minutes. Each treatment (dose) was testedusing five test cages, i.e. on a total of 250 female A. gambiae. Thelimits of the boxes indicate the twenty-fifth and seventy-fifthpercentiles, the solid line in the box is the median, the capped barsindicate the tenth and the ninetieth percentiles, and data pointsoutside these limits are plotted as circles.

Ternary mixtures were also tested at a total concentration of 2 nmol/cm²and achieved effects that were similar to those obtained with the binarymixtures. (Results not shown in Table 5 and FIG. 2).

Example 8 Spatial Repellent Effect of Compound Carvacrol

Carvacrol was tested in a wind tunnel (60×60×120 cm) in order to testfor inhibition of the attractiveness of dirty socks, which are otherwisevery attractive to the malaria mosquito A. gambiae. Experiments wereconducted at 25° C. and 80% relative humidity at a wind speed of 0.5m·s⁻¹ during the last 6 hours of scotophase. Dim fluorescent light (4Lux) was provided from above (Philips TLD, 32 Watts at 36 KHz). A tube(55 mm inner diameter) was covered at its downwind end with mosquitonetting which was treated with 38 or 57 μg DEET/cm² or 38 or 76 μgcarvacrol/cm² or the solvent (ethanol) alone (control). Groups of 30 A.gambiae females were released from a release cage at the downwind end ofthe wind tunnel and their landing behaviour on the mosquito netting wasrecorded with a high definition video camera. The number of mosquitoeslanding on the mosquito netting over 5 minutes was counted. Whereaslandings by A. gambiae females on the netting downwind of the sock washigh in controls, the mosquito netting treated with either DEET orcarvacrol clearly inhibited A. gambiae females from landing on thedownwind end of the tube containing the dirty sock (Table 6).

TABLE 6 Spatial Repellence Effect of Compound Carvacrol Compared to DEETMean Landings/5 Compound μg/cm² nmol/cm² minutes Control 0 0 24.6 DEET38 200 0 DEET 57 300 0 Carvacrol 38 250 0 Carvacrol 76 500 0

Example 9 Field Testing of Spatial Repellent Effects of Compounds

To test the effects of repellent compounds or compositions disclosedherein on mosquitoes in a controlled environment resembling actualliving quarters and conditions, field trials with standardizedexperimental huts were conducted at Kainji Dam in North Central Nigeria.

Six such huts were built in a straight line, and the distance betweenhuts was 11 meters. These huts follow a traditional square design, thewalls are built with concrete blocks that are subsequently plasteredwith cement, each wall has a window. The ceiling is plywood and the roofis thatched with eaves that are open, thereby allowing free flow of airinto and out of the hut, and thus entry and exit of mosquitoes. There isa door for entry and exit. Two opposing sides of each hut have windowexit traps and screened verandas to capture mosquitoes leaving via thewindows or eaves. Each hut is raised off the ground on a concreteplatform and surrounded by a moat filled with water in order to preventants from entering. Each hut is fitted with a mattress and an untreatedbed net for sleepers. These huts are similar in design and concept tothose used previously elsewhere for studying mosquito behavior andmeasuring the efficacy of various mosquito control products orintervention methods in Tanzania and Gambia. For design parameters referto WHOPES 2005. The huts are adjacent to a tributary of the River Nigerat a distance of 10 km from the Kainji Dam site and face rice fieldsknown to harbor permanent mosquito breeding sites. The mosquitoesendemic to this area of Nigeria are highly endophillic, that is, theyrest mainly indoors.

Human volunteers that participated as sleepers in this field trial wereprovided with anti-malarial chemical prophylaxis on a weekly basis, andthe protocols used herein were reviewed and approved by the ResearchEthics Review Committee of the Nigerian Institute of Medical Research.Six male adult volunteers, aged between 25 and 35 years old, wererecruited to sleep in the huts described above. Sleepers were allocatedto huts randomly for each series of trials, and this randomizationprocess occurred nightly. The volunteers entered the huts by 21:00 eachnight and remained inside the huts until 06:30 the following morning.Each morning, the windows were closed and the exit traps blocked with apiece of cloth to prevent mosquitoes that entered from exiting. Thetraps were then put in a freezer at −20° C. to kill all the mosquitoesbefore being emptied for identification. By means of a sucking aspiratorthe verandas and rooms were visually searched for live mosquitoes andany insects found were collected in labelled cups. A 10-minute searchwas conducted for each hut (room and veranda traps). The exit traps wereemptied into a labelled cup and all the mosquitoes placed in the freezerat −20° C. for about 30 minutes before morphological identification.

The Mosquito Magnet trap Model-X (MM-X trap made by the firm formerlyknown as American Biophysics (ABC), USA) is a 12V battery operatedcounter-flow trap that was used as the repellent dispersing device toevaluate the efficacy of repellent compounds or compositions disclosedherein. Four compounds, carvacrol, cumin alcohol, ethyl cinnamate, andbutyl cinnamate, were evaluated in comparison to DEET as anonproprietary conventional repellent in a first experiment. Thechemical compounds were provided in liquid form: carvacrol (98%), butylcinnamate (98%), ethyl cinnamate (98%), cumin alcohol (97%) and DEET(97%). 1 ml of each compound was introduced into a 2.5 ml open vial of1.2 cm diameter. This resulted in a surface area of 1.13 cm² in thevial. Each vial was then clipped onto a string and placed in the centralMM-X trap compartment. A second experiment compared the effect ofcombination of two compounds, each MM-X trap contained 2 vials, eachwith a single chemical (0.5 ml volume). A hut with a sleeper with anMM-X trap baited with human odour from a nylon sock was used as control.

The trap was hung 20 cm above the hut floor inside the hut near themattress of each hut with a human volunteer sleeping in the hut. Trapswere switched on by 21:00 each evening to allow for product release andremoved each morning by 06:30. New vials were prepared for each day. Theexperiment was designed using Latin Square approach with randomizationbased on 6 huts×12 nights array that was balanced to control for anycarry over effect of treatments. Spatial repellent effects of productswere assessed based on mosquito counts in the exit traps (ExTr in FIGS.3 and 4; i.e. induced exophily), counts on the walls inside the huts andin the MM-X traps. Numbers of mosquitoes captured in window exit trapswere compared in each experiment using a generalized linear model with aquasibinomial response followed by a Post-hoc Tukey test (in R V3.0.1).Findings were checked for carry-over effects of a product tested withina hut the night before.

The 144 hut-night collections (24 nights×6 huts) yielded 1466 (38%)Anopheles spp., 2231 (53%) Culex spp. while the remaining was Mansoniaspp. The Anopheles population showed a predominance of Anopheles gambiaes.l. consisting of 85% Anopheles gambiae s.s and 15% Anophelesarabiensis.

The spatial repellent effect of each product was expressed as thepercentage of mosquitoes counted in the exit traps (induced exophily),on the walls and in the trap (FIGS. 3 and 4). Carvacrol had a spatialrepellent effect similar to that of DEET (P>0.05) and significantlyhigher in terms of induced exophily on Anopheles spp. and Culex spp.than cumin alcohol, ethyl-cinnamate, butyl-cinnamate and the control(P<0.001; FIGS. 3 A and 3 B). The repellent effect of cumin alcohol,ethyl-cinnamate, butyl-cinnamate was significantly lower than that ofDEET (P<0.001; FIGS. 3 A and 3 B). The induced exophily resulted inreduced mosquito counts on the inside walls of the huts for carvacroland DEET, much lower than on the walls in the huts with cumin alcohol,ethyl-cinnamate, butyl-cinnamate and the control (FIGS. 3 A and 3 B).

Based on the above results and those presented in Example 7, thepossible additive effects of combinations of compounds were tested. Thetraps were set up with combinations of carvacrol+cumin alcohol,carvacrol+ethyl-cinnamate and carvacrol+butyl-cinnamete compared to DEET(positive control). MM-X traps baited with methanol and empty traps wereused as negative controls (FIGS. 4 A and 4 B). The experiment was againdesigned using the Latin Square design with randomization based onproduct×hut×night as indicated above.

The binary combination of carvacrol+cumin alcohol produced the highestrepellent effect in terms of induced exophily on Anopheles and Culexspp. compared to the other binary combinations (P<0.05; FIGS. 4 A and 4B), but was not different to DEET (P>0.05).

The described spatial repellent effects were achieved with release ratesof approximately 1-10 mg amounts of products per night. Effects of thespatial repellents on Culex spp. were similar to those recorded forAnopheles spp., as can be seen by comparing FIG. 3A with FIG. 3B andFIG. 4 A with FIG. 4 B.

Example 10 Effects of Repellent Compounds on Other Blood-FeedingArthropods—Sand Flies

To test the effects of repellent compounds or compositions disclosedherein on other blood-feeding arthropods, a warm body repellence assay(Krober et. al. (2010). An In Vitro Assay for Testing MosquitoRepellents Employing a Warm Body and Carbon Dioxide as a BehavioralActivator. Journal of the American Mosquito Control Association 26(4):381-386] was conducted using the sand fly Lutzomyia longipalpis(Diptera: Psychodidae), the principal vector of Leishmania (orleismaniasis) in the Americas.

To conduct a repellence assay with the sand fly, pure compounds orethanol as negative control were applied to the warm body at a dose ofabout 3.8 μg/cm2 and tested for 2 minutes as described in Example 6.Fifty L. longipalpis sandflies (male:female ratio 1:1) of 3-7 days oldwere tested during the scotophase under conditions described for A.gambiae in Example 6. The test cages were lined with filter paper.Reduction in the number of landings by L. longipalpis sand fly femaleson a warm body treated with different test products compared to thecontrol with solvent (ethanol p.a.) alone was used to evaluaterepellence. Tests were conducted in complete darkness using IR LEDs toilluminate the warm body. Landings counted during 2 minutes werenormalized by experimental day, cage, and number of females by a mixedlinear model before testing for differences. Carvacrol and cuminic acidpresented at 20 and 200 nmol/cm² were as effective as DEET at 20nmol/cm² at repelling L. longipalpis sand flies from the warm body(Table 7).

TABLE 7 Repellent Effect of Candidate Compounds on L. longipalpisCompared to DEET Test Concentration Median of Difference to DEET product(nmol/cm²) landings at 20 nmol/cm² Control — 48 *** DEET 20 6 Referencelevel 200 5 ns butyl 20 49 *** cinnamate 200 40 * carvacrol 20 23 ns 2001 ns cuminic 20 16 ns acid 200 13 ns * P ≦ 0.1; ** P ≦ 0.01; *** P ≦0.001; ns, not significantly different to DEET

Example 11 Effects of Repellent Compounds on Other Blood-FeedingArthropods—Ticks

The products were tested at a dose of 10 μg/cm² on Ixodes ricinus(Ixodidae) nymphs according the protocol described in Krober, T. et al.(2013). A standardised in vivo and in vitro test method for evaluatingtick repellents. Pesticide Biochemistry and Physiology 107: 160-168]withthe following modifications: The test products (dissolved in ethanol)were allowed to evaporate for 40s (as in the warm body test withmosquitoes) and the warm glass plates were used up to 250 s afterapplication of the test products for the repellence assay. Fourrepetitions were made with carvacrol with 3, 4, 4 and 5 nymphs, i.e.with a total of 16 I. ricinus nymphs, of which in total 75% (12) wereaffected in that they either dropped off or walked down the warm glassplate.

The mixture of carvacrol+cumin alcohol+butyl-cinnamate was tested in tworepetitions with 6 and 7 nymphs, i.e. with a total of 13 I. ricinusnymphs, of which 100% were affected in that they either dropped off orwalked down the warm glass plate. Of 12 Ixodes ricinus tested in thecontrol (ethanol only) only one tick (8%) was affected (Table 8).

TABLE 8 Repellent Effect on Arachnid Compound % affected ticks control 8carvacrol 75 carvacrol + cuminic alcohol + 100 butyl cinnamate

Concluding Remarks

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe invention are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein is intended merely to betterilluminate the present invention and does not pose a limitation on thescope of the invention otherwise claimed. No language in the presentspecification should be construed as indicating any non-claimed elementessential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using “consisting of” or “consisting essentially of” language.When used in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

1. A method of repelling an insect and/or an arachnid, the methodconsisting essentially of applying a composition to an animal, a human,a substrate and/or a location, the composition consisting essentially ofan insect repelling amount and/or an arachnid repelling amount of acarvacrol compound of formula I and an insect repelling amount and/or anarachnid repelling amount of a cumin compound of formula II, whereinformula I is

wherein R¹ is H, OH, ═O, a halogen, an optionally substituted alkyl, analkoxy; wherein formula II is

wherein R² is H, OH, ═O, a halogen, a carbonyl group (CHO), a carboxylgroup (OOH), or an optionally substituted alkyl, or an alkoxy; whereinapplication of the composition to the animal, the human, the substrateand/or the location repels the blood-feeding arthropod from the animal,the human, the substrate and/or the location.
 2. The method according toclaim 1, wherein application of the composition is by topicaladministration.
 3. The method according to claim 1, wherein the insectis a blood-feeding insect.
 4. The method according to claim 3, whereinthe blood-feeding insect is a mosquito or sand fly.
 5. The methodaccording to claim 1, wherein the arachnid is a blood-feeding arachnid.6. The method according to claim 5, wherein the blood-feeding arachnidis a tick.
 7. The method according to claim 1, wherein the location is aplant or group of plants, a particular area of land, or a man-madestructure.
 8. The method according to claim 7, wherein the man-madestructure is a commercial building, a house, a shed, a livestockmaintenance area, other physical structure, or any part thereof.
 9. Amethod of reducing bites of a blood-feeding arthropod in an animal or ahuman, the method consisting essentially of applying a composition tothe animal or the human, the composition consisting essentially of ablood-feeding arthropod repelling amount of a carvacrol compound offormula I and a blood-feeding arthropod repelling amount of a cumincompound of formula II, wherein formula I is

wherein R¹ is H, OH, ═O, a halogen, an optionally substituted alkyl, analkoxy; wherein formula II is

wherein R² is H, OH, ═O, a halogen, a carbonyl group (CHO), a carboxylgroup (OOH), or an optionally substituted alkyl, or an alkoxy; whereinapplication of the composition repels the blood-feeding arthropod fromthe animal or the human, thereby reducing bites of blood-feedingarthropods.
 10. The method according to claim 9, wherein application ofthe composition is by topical administration.
 11. The method accordingto claim 9, wherein the blood-feeding arthropod is a blood-feedinginsect or a blood-feeding arachnid
 12. The method according to claim 11,wherein the blood-feeding insect is a mosquito or sand fly.
 13. Themethod according to claim 11, wherein the blood-feeding arachnid is atick.
 14. A method of reducing an infestation of a blood-feedingarthropod in a location, the method consisting essentially of applying acomposition to the location, the composition consisting essentially of ablood-feeding arthropod repelling amount of a carvacrol compound offormula I and a blood-feeding arthropod repelling amount of a cumincompound of formula II, wherein formula I is

wherein R¹ is H, OH, ═O, a halogen, an optionally substituted alkyl, analkoxy; wherein formula II is

wherein R² is H, OH, ═O, a halogen, a carbonyl group (CHO), a carboxylgroup (OOH), or an optionally substituted alkyl, or an alkoxy; whereinapplication of the composition to the location repels the blood-feedingarthropod from the location, thereby reducing the infestation.
 15. Themethod according to claim 14, wherein application of the composition isby spraying.
 16. The method according to claim 14, wherein theblood-feeding arthropod is a blood-feeding insect or a blood-feedingarachnid
 17. The method according to claim 16, wherein the blood-feedinginsect is a mosquito or sand fly.
 18. The method according to claim 16,wherein the blood-feeding arachnid is a tick.
 19. The method accordingto claim 14, wherein the location is a plant or group of plants, aparticular area of land, or a man-made structure.
 20. The methodaccording to claim 19, wherein the man-made structure is a commercialbuilding, a house, a shed, a livestock maintenance area, other physicalstructure, or any part thereof.